COMPOSITIONS AND METHODS FOR CaMKII INHIBITORS AND USES THEREOF

ABSTRACT

Embodiments herein generally relate to methods, compositions and uses of CaMKII inhibitors. Other embodiments relate to methods, compositions and uses of agents that target CaMKII. Yet further embodiments relate to compositions, methods and uses of CaMKIIN-derived molecules and other CaMKII inhibitor molecules that inhibit autonomous CaMKII activity. In accordance with these embodiments, compositions that inhibit autonomous CaMKII activity may be used for treating conditions causing neuronal cell death, for treating cancer or for treating neurodegenerative disorders.

RELATED APPLICATIONS

The instant application is a continuation-in-part application thatclaims the benefit of U.S. non-provisional application Ser. No,12/679,459 filed under 35 USC 371 that claims the benefit of PCTUS2008/77934 application filed Sep. 26, 2008 that claims the benefit ofProvisional Patent Applications Ser. No. 60/980,766, filed Oct. 17, 2007and Ser. No. 60/975,329, filed Sep. 26, 2007. These applications areincorporated herein by reference in their entirety for all purposes.

FIELD

Embodiments herein generally relate to methods, compositions and uses ofcalcium/calmodulin (CaM)-dependent protein kinase II (CaMKII)inhibitors. In addition, embodiments herein generally relate to methods,compositions and uses of agents that target CaMKII. Other embodimentsrelate to compositions, methods and uses of CaMKIIN-derived moleculesand other molecules that inhibit autonomous CaMKII activity, of use totreat neurodegenerative disorders, neuronal cell death or subjects afteracute insult.

BACKGROUND

CaMKII is a multifunctional protein kinase known for its critical rolein learning and memory. CaMKII is highly expressed in the brain, but atleast one of its four isoforms (α, β, γ and δ) has been found in everycell type examined to date. Numerous cellular functions of CaMKII havebeen described, both in and outside the nervous system. Some of thesefunctions include regulation of various ion channels, gene expression,cell cycle/proliferation control, apoptotic and excitotoxic cell deathcell morphology and filopodia motility. CaMKII is also implicated inregulation of insulin secretion, although studies suggesting this werelargely based on KN inhibitors that also affect Ca2+-channels requiredfor secretion.

CaMKII forms multimeric holoenzymes and a CaM-dependent inter-subunitauto-phosphorylation at T286 renders the kinase active even afterdissociation of Ca2+/CaM. T286 is located in the regulatory region; itsphosphorylation relieves auto-inhibition by preventing binding of theregion around T286 to the T-site, which is adjacent to the substratebinding S-site. The Ca2+-independent or autonomous activity has beenregarded as a form of “molecular memory”, and is important in severalneuronal functions of the kinase. Additionally, T286 phosphorylationtraps CaM on CaMKII, and regulates CaMKII binding to other proteins,such as syntaxin, densin-180, NR1, NR2A, NR2B, and F-actin. Among theother auto-phosphorylation sites, functions of T305/306 are understoodbest. T305/306 auto-phosphorylation can occur in an intra-subunitreaction, blocks CaM binding, accelerates CaMKII dissociation fromsynaptic sites, and also plays a role in learning.

SUMMARY

Embodiments of the present invention provide for methods, compositionsand uses of calcium/calmodulin (CaM)-dependent protein kinase II(CaMKII) inhibitors protein CaM-KIIN. Certain embodiments concerncompositions including at least a portion of the CaMKII inhibitorprotein CaM-KIIN. Some embodiments concern compositions of about 5, toabout 10, to about 20, to about 25, to about 30, to about 40, to about50, to about 100 or more consecutive amino acids of CaM-KIIN. Inaccordance with these embodiments, CaM-KIIN molecules or fragmentsthereof can be administered as a composition alone, linked to atransporter agent, associated with microparticles, or other deliverysystem. In other embodiments, a peptide derived from a CaM-KIIN moleculemay include one or more amino acids that differ from native CaM-KIIN. Inaccordance with these embodiments, amino acid changes within a peptidederived from CaM KIIN may increase, decrease or maintain potency ofCaMKII inhibition.

Other embodiments herein can concern a composition of a fragment,portion or truncated form of CaM-KIIN linked to cell penetratingsequences. For example, a ‘tat,’ or an ‘ant’ sequence (tat: YGRKKRRQRRR,SEQ ID NO:15; ant: RQIKIWFQNRRMKWK, SEQ ID NO:16), meristyl-group,palmityl-group or combination thereof, can be covalently ornon-covalently associated with a portion of CaM-KIIN. Other embodimentsherein concern a modified portion of CaM-KIIN made cell penetrating or amodified CaM-KIIN molecule, derivative of or fragment thereof designedto have a longer half-life or increased potency for inhibition ofCaMKII. In certain embodiments, the fragment or portion of CaM-KIIN canbe 5 consecutive amino acids or more of SEQ ID NO:1. In otherembodiments, compositions contemplated herein can include one or morepeptides derived from SEQ ID NO:1 including 30 or less amino acids inlength, 25 or less amino acids in length, 20 or less amino acids inlength, or 15 or less amino acids in length.

In yet other embodiments, CaMKII inhibitor peptides (CN-related peptidesor CN peptides) can be used in compositions and methods described hereinto regulate CaMKII activity in a subject or for research in a laboratorysetting. CN-related peptides can also include peptides optimized forCaMKII potent and/or selective inhibition over other kinases. Inaccordance with these embodiments, a CaMKIIN-derived peptide can includeCN19a2v (SEQ ID NO 23) or CN19o (SEQ ID NO 24) described herein. Forexample, compositions including CN19o can be used to completely orselectively to inhibit CaMKII activity in a subject to treat forexample, neurological disorders, to modulate memory/learning behavior orother functions of CaMKII in a subject. Alternatively, inhibitors ofsome embodiments can be used to identify additional functions of CaMKIIin a research setting in order to further assess treatments related toconditions disclosed herein. It is contemplated that inhibitors ofCaMKII may be used to modulate a neuronal function by an on/offmechanism or a regulatory mechanism to reduce or increase CaMKIIactivity (not completely turn on or off the activity) based on need.

Other embodiments concern compositions and methods for reducing neuronalcell death in a subject. In accordance with these embodiments,compositions contemplated herein can be used to treat a subject havingor at risk of developing a neurodegenerative disease. In certainembodiments, compositions disclosed herein can be used to decreaseprogression of neurodegenerative disease or disorders. Disorders caninclude, but are not limited to, stroke, ischemia, traumatic braininjury, Alzheimer's disease, Parkinson's disease, drug addiction, spinalcord injury, regulation of insulin secretion, or cancer. In otherembodiments, compositions and methods contemplated herein can be used totreat a subject having a brain injury for example, a traumatic braininjury. In some embodiments, compositions and methods herein can be usedto reduce, inhibit or prevent toxic effects of excessive excitatoryneurotransmitters. For example, one exemplary neurotransmitter may beglutamate. In certain embodiments, reduction of toxic effects ofexcessive excitatory neurotransmitters may be at least a 10%, or atleast a 15%, or at least a 25%, or at least a 50%, or more reduction inside effects due to toxic effects of excessive excitatoryneurotransmitters. In some particular embodiments, side effects caninclude, but are not limited to, cell death, neuronal activation and/orneuronal translocation. In certain embodiments, a compositioncontemplated for reducing neuronal cell capacity can include 5, 10, 20,25, 30, 40, 50, 100 or more consecutive amino acids of CaM-KIIN proteinalone, or linked to or associated with a cell-transporter/penetratingagent.

In other embodiments, CaMKII inhibitor peptides are optimized by meansknown in the art. For example, CN peptides can be made to have increasedcell-penetrating capabilities, increased potency to inhibit CaMKIIand/or increased stability. In certain embodiments, CN peptides can bemade cell-penetrating where such cell-penetrating CN peptides can beprotective from glutamate excitotoxicity even when applied after aninsult occurs to a subject. In other embodiments, autonomous CaMKIIactivity can be a target for post-insult neuro-protection, for example,administering CaM-KIIN molecules or fragments or other molecules thattarget autonomous CaMKII activity to a subject in need of such atreatment. In yet other embodiments, mutant forms of the inhibitoryregion of CaM-KIIN can be derived with increased potency of inhibitionand used in compositions and methods disclosed herein. In someembodiments, a composition of a peptide including 25% or moreconsecutive amino acids of SEQ ID NO:1, CN21, alone or fused to acell-penetrating agent is contemplated of use herein. In otherembodiments, a composition of a peptide including 25% or moreconsecutive amino acids of SEQ ID NO:2, CN19, alone or fused to acell-penetrating agent is contemplated of use herein.

It is contemplated that administration to a subject in need thereof ofany of the disclosed compositions may be used alone or in combinationwith other agents used to treat conditions herein (e.g. neuronal celldeath, acute insult, traumatic brain injury, stroke, neurodegenerativeconditions).

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments herein. Theembodiments may be better understood by reference to one or more ofthese drawings alone or in combination with the detailed description ofspecific embodiments presented.

FIGS. 1A-1D represent an exemplary amino acid sequences of a portion ofCaM-KIIN, SEQ ID NO:1, (FIG. 1A). 1B represents an exemplary histogramof CaMKII activity measured in the presence of indicated portions ofCaM-KIIN. 1C represents exemplary kinases tested for inhibition ofactivity in the presence of various agents. 1D represents exemplaryeffects of CN27 and CN21 on CaMKII isoforms.

FIGS. 2A-2C represent exemplary effects of CN peptides onphosphorylation of two peptide substrates. 2A represents effects of CNpeptides on phosphorylation of peptide substrate syntide2. 2C representsa competition assay between CN21 and AC2.

FIGS. 3A-3C represent interactions of CaM-KIIN and CN21 with respect toCaMKII T-site. 3A represents a molecular model of a CaMKII kinase domainwith some of the proposed associating molecules identified. 3Brepresents a histogram of a comparison to GFP-CaMKIIα wild type. 3Crepresents a Western blot analysis of Ca²⁺/CaM-induced CaMKII binding toimmobilized GST-NR2B-c.

FIG. 4 represents an exemplary plot representing CaM dissociation fromCaMKII.

FIGS. 5A-5D represent exemplary results of whether CaMKIIautophosporylation can be blocked by CaM-KIIN or CN21. 5A-5C representexemplary gels indicating that CaM-KIIN and CN21 block substrate- andT305 but not T286 auto-phosphorylation. 5A represents an exemplary gelillustrating that CN peptides blocked CaMKII substrate. 5B represents anexemplary gel of a time course of CaMKII auto-phosphorylation. 5Crepresents a slot-blot analysis (left panel) was performed forquantification represented in a histogram plot (right panel). 5Drepresents an exemplary mobility shift gel.

FIGS. 6A-6B represent an exemplary construct of a CaMKIIN derivedmolecule, CN21 linked to tat, termed tatCN21. 6A illustrates the effectof tatCN21 on a panel of different kinases. 6B illustrates an exemplaryWestern blot of CaMKII binding.

FIGS. 7A-7C represent motility of hippocampal neurons. 7A representsexemplary images of the same dendrite area before and 20 min afteraddition of tatCN21, at different times of acquisition as indicated. 7Brepresents average of A images in pseudo color visualize motility in alarger area (110×148 μm). 7C represents quantification of a change inmotility after application of tatCN21 or tatRev control based on A imageaverage projections.

FIG. 8 represents an exemplary histogram illustrating a portion of aCaM-KIIN molecule and its effects on glucose induced insulin secretion.

FIGS. 9A-9G illustrate ant and tat fusion to an inhibitor peptide andtheir direct binding to calmodulin. 9A illustrates that the ant and tatsequences fused to the N-terminus of CNs. 9B represents an exemplaryhistogram that illustrates extent of CaMKII inhibition by antCN27 andthe reverse sequence control antRev depended on the CaM concentration.9C represents an exemplary histogram illustrating tatCN21 inhibitedCaMKII-mediated AC3 phosphorylation at all CaM concentrations, while thereverse and scrambled sequence controls tatRev and tatScr had no effectcompared to assays without tat peptide. 9D represents an exemplary plotof kinase activity in the presence of various agents. 9E representsexemplary binding of biotinylated CaM to peptides immobilized by slotblot. 9F represents an exemplary blot overlay assay in the presence orabsence of various peptides. 9G represents an exemplary plot offluorescence of TA-CaM in the presence or absence of antCN27, ant ortatCN21.

FIG. 10 illustrates exemplary gel analysis of a differential effect oftat- and ant-CN fusion peptides on CaMKII substrate- andauto-phosphorylation.

FIGS. 11A-11E represent exemplary histograms of neuronal cell deathmeasured by LDH release. In this exemplary method, glutamate was used toinduce neuronal cell death and a construct was analyzed to examinewhether it was capable of attenuating neuronal cell death before andafter glutamate insult (11A-11E). 11D represents an exemplary histogramof 7DIV cortical up to 6 hours after insult in the presence or absenceof tatCN21. 11E represents an exemplary histogram (left panel)illustrating siRNA targeting CaMKIIα (siα) and expression of CaMKII in10DIV hippocampal neurons as indicated by western-blot (right panel).

FIGS. 12A-12D represents exemplary data of KN93 and tatCN21 effects onCaMKII. 12A represents an exemplary histogram of tatCN21 and KN93effects on calcium and calmodulin stimulated CaMKII activity. 12Brepresents an exemplary blot representing self-association in vitro inthe presence or absence of KN93 or tatCN21. 12C represents an exemplaryWestern analysis measuring binding to the immobilized NR2B subunit ofthe NMDAR in the presences or absence of tatCN21, KN93, or anon-specific kinase inhibitor, H7. TatCN21 and KN93. 12D represents anexemplary table summarizing effects of tatCN21 and KN93 on CaMKIIactivity, NR2B binding, or self-association.

FIGS. 13A-13B represent exemplary histograms of overexpression of T286Aand glutamate induced cell death compared with overexpression ofCaMKIIa. 13B represents an exemplary histogram of LDH activity in thepresence or absence of CaMKII overexpression

FIGS. 14A-14C illustrates exemplary gel analysis of a minimal inhibitoryregion of CaM-KIIN for its retention of CaMKII specificity. 14Aillustrates an exemplary gel measuring phosphorylation activity in thepresence or absence of CN21a. 14B represents an exemplary gel measuringphosphorylation activity in the presence or absence of bacteriallyexpressed GST-NR2B-c (containing C-terminal NR2B amino acid 1,120 to1,482). 14C represents an exemplary gel measuring autophosphorylationactivity using phosphospecific antibodies.

FIGS. 15A-15D represents affects of various peptides on CaMKII activity.15A represents, CN19, one minimal inhibitor region of CaM-KIIN. Arrowsindicate truncations. 15B-15D represent exemplary histograms of CN19 andfurther truncations and their inhibitory potency determined by effectson CaMKII activity.

FIGS. 16A and 16B represents various construct effects on CaMKI activity(**), tatCN21,as well as, tat alone and other tat fusions, CN27, 21, and19 alone. 16A represents an exemplary histogram of these effects. 16Brepresents an exemplary plot of a dose response of CaMKI inhibition bytatCN21, tat, and CN21.

FIGS. 17A-17B represent a summary of the mutational analysis of CN19 andeffects on potency of CaMKII inhibition. 17B represents a summary ofspecific mutations of CN19 on potency of CaMKII inhibition.

FIGS. 18A-18C represent exemplary data of several point mutations to anArginine (R) and their effect on potency of CaMKII inhibition. 18Arepresents an exemplary histogram of percent of control in the presenceof various mutations of CN19-a3. 18B represents an exemplary histogramof a mutant, CN19a2-m1 and its effects on inhibitory potency. 18Cillustrates additional mutations made in the combination mutant series.

FIGS. 19A-19B represent exemplary histograms of CN19 mutations at S12and effects of these mutations on CaMKII inhibition. 19A illustrates S12mutations to potency of CaMKII inhibition. 19B represents an exemplaryhistogram of the effects S12 mutations in a CN19a2 background on CaMKIIactivity.

FIGS. 20A-20C represents exemplary histograms of potency of combinationmutants of CN19. 20A represents an exemplary histogram of the effects ofcombination mutants aX, a3, a4, and a5 on potency of CaMKII inhibitioncompared to CN19 wild type. 20B represents an exemplary histogram of theeffects on potency of CaMKII inhibition of other combination mutants athigher concentration. 20C represents an exemplary histogram of theeffects on potency of CaMKII inhibition of P4 (see FIG. 17B).

FIGS. 21A-21C represents exemplary histograms of the effects on potencyof CaMKII inhibition by combination mutants II. (21A and 21B). 21Crepresents an exemplary histogram of the effects on potency of CaMKIIinhibition by an a2 mutant, an a3 mutant, or an 14A mutant and othermutants as indicated (see FIG. 17B).

FIGS. 22A and 22B represent exemplary histograms demonstratingpost-translational modification of CN19 reuces potency of CaMKIIinhibition, 22A represents percent enzyme activity of CN19 compared tocontrols and 22B represents CAMKII activity in control versuspost-translationally modified CN19.

FIGS. 23A and 23B represents an exemplary histograms demonstratingCAMKII activity in the presence of various mutants of CN19.

FIGS. 24A-24C represent A. Sequences of various CN19 mutants, and B. Anexemplary histogram plot of various CN19 mutants and their effect onCaMKII inhibition and C. An exemplary plot of various CN19 mutants invarious combinations and their comparitory effect on CaMKII inhibition

FIGS. 25A and 25B represent an exemplary plot of a potent CaMKIIinhibitor (A) and an exemplary histogram plot of the inhibitor toinhibit various enzymatic activities compared to effects on CaMKII.

FIG. 26 represents an exemplary plot and table summarizing experimentsusing various CN19 peptide sequences and their effects on CaMKII versusCaMKI.

DEFINITIONS

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, vessel can include, but is not limited to, test tube,mini- or micro-fuge tube, channel, vial, microtiter plate or container.

As used herein the specification, “subject” or “subjects” may includebut are not limited mammals such as humans or mammals, domesticated orwild, for example dogs, cats, ferrets, rabbits, pigs, horses, cattle,zoo animals, or wild animals.

As used herein, “about” can mean plus or minus ten percent.

As used herein “modulate” can mean to increase or up-regulate activityor function; or to decrease or down-regulate activity or function of atarget molecule (e.g. CaMKI or CaMKII).

An “antibody” as used herein refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment. Theterm “antibody” also includes “humanized” antibodies and even fullyhuman antibodies that can be produced by phage display, gene andchromosome transfection methods, as well as by other means. This termalso includes monoclonal antibodies, polyclonal antibodies, multivalentantibodies, multispecific antibodies (e.g., bispecific antibodies).

“Polyclonal antibodies” are generated in an immunogenic response to aprotein having many epitopes. A composition (e.g., serum) of polyclonalantibodies thus includes a variety of different antibodies directed tothe same and to different epitopes within the protein. Methods forproducing polyclonal antibodies are known in the art (see, e.g., Cooperet al., Section III of Chapter 11 in: Short Protocols in MolecularBiology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New York,1992, pages 11-37 to 11-41).

A “monoclonal antibody” is a specific antibody that recognizes a singlespecific epitope of an immunogenic protein. In a plurality of amonoclonal antibody, each antibody molecule is identical to the othersin the plurality. In order to isolate a monoclonal antibody, a clonalcell line that expresses, displays and/or secretes a particularmonoclonal antibody is first identified; this clonal cell line can beused in one method of producing the antibodies of the present invention.Methods for the preparation of clonal cell lines and of monoclonalantibodies expressed thereby are known in the art (see, for example,Fuller et al., Section II of Chapter 11 in: Short Protocols in MolecularBiology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New York,1992, pages 11-22 to 11-11-36).

DETAILED DESCRIPTION

In the following sections, various exemplary compositions and methodsare described in order to detail various embodiments of the invention.It will be obvious to one skilled in the art that practicing the variousembodiments does not require the employment of all or even some of thespecific details outlined herein, but rather that sequences chosen,proteins selected, samples, concentrations, times and other specificdetails may be modified through routine experimentation. In some cases,well-known methods or components have not been included in thedescription.

CaMKII is a multifunctional protein kinase best known for its criticalrole in learning and memory. Ca2+/calmodulin-dependent protein kinasesII or CaM kinases II are serine/threonine-specific protein kinases thatare regulated by the calcium/calmodulin complex. CaMKII is involved inseveral signaling cascades. CaMKII is highly expressed in the brain, butat least one of its four isoforms (α, β, γ and δ) has been found inevery cell type examined. Numerous cellular functions of CaMKII havebeen described, both in and outside the nervous system. These includeregulation of various ion channels, gene expression, cellcycle/proliferation control, apoptotic and excitotoxic cell death, cellmorphology and filopodia motility. CaMKII has also been implicated inregulation of insulin secretion and in several signaling cascades,positive T-cell selection, and CD8 T-cell activation.

Misregulation of CaMKII has been demonstrated as linked to Alzheimer'sdisease, Angelman's syndrome, and heart arrhythmia. Therefore,regulation of this complex multifunctional kinase can be important inregulating progression or reducing symptoms of these indications in asubject.

CaMKII forms multimeric holoenzymes and a CaM-dependent inter-subunitauto-phosphorylation at T286 renders the kinase active even afterdissociation of Ca2+/CaM. Phosphorylation of T286, which is located inthe regulatory region, relieves auto-inhibition by preventing binding ofthe region around T286 to the T-site, which is adjacent to the substratebinding S-site (see kinase domain model in FIG. 3A). The subsequentCa2+-independent or autonomous activity has been regarded as a form of“molecular memory”, and it is important in several neuronal functions ofthe kinase. Additionally, T286 phosphorylation traps CaM on CaMKII, andregulates CaMKII binding to other proteins, such as syntaxin,densin-180, NR1, NR2A, NR2B, and F-actin. Other auto-phosphorylationsites previously examined are functional consequences of T305/306phosphorylation. T305/306 auto-phosphorylation can occur in anintra-subunit reaction, blocks CaM binding, accelerates CaMKIIdissociation from synaptic site, and also plays a role in learning.

CaMKII inhibitors such as KN62, KN93, and peptides derived from theautoinibitory region of CaMKII, such as AIP or AC3-I, are useful toolsfor examining functions of the kinase. However, the KN drugs cannotdiscriminate between CaMKII and CaMKIV and they inhibit voltage-gated K+and Ca2+ channels. Moreover, the KN drugs interfere competitively withactivation by CaM and thus do not inhibit autonomous activity of thekinase. The CaMKII-derived peptide inhibitors are widely believed to bemore specific. However, such peptides also inhibit other CaM-dependentkinases as well as protein kinase A, and their potency is low. Thus,embodiments herein concern a natural CaMKII inhibitor protein, CaM-KIINbecause it may provide a promising alternative to other inhibitors, asit potently inhibits only CaMKII.

CaM-KIIN is a natural CaMKII inhibitor protein expressed in brain, whereCaMKII is also most abundant (constituting up to 2% of total protein).Regulation of CaMKII activity is known to be required for controllingforms of synaptic plasticity underlying higher brain functions such aslearning and memory. CaM-KIIN can interfere with all of CaMKIIregulatory mechanisms: It is competitive with GluN2B binding andefficiently inhibits CaMKII activity, as well as T305/306auto-phosphorylation. But, it only mildly reduces T286auto-phosphorylation, while effectively blocking autonomous activity. Incontrast to CaMKII, which is enriched at dendritic spine synapses,CaM-KIIN is restricted to the dendritic shaft in a subject, suggestingspecific local control of CaMKII regulation.

Some embodiments disclosed herein concern compositions that can includea region of CaM-KIINα that retains full potency and specificity ofCaMKII inhibition (referred to as CN21) (the homologous CaM-KIINβ regiondiffers at one residue only). In accordance with these embodiments, CN21efficiently blocked substrate- and T305 auto-phosphorylation of CaMKII,but only mildly affected T286 auto-phosphorylation. Identification ofthe T-site as the CaM-KIIN interaction site on CaMKII provided twomechanisms for this novel differential inhibitor effect: CaM-KIIN wascompetitive with the region around T286, and strengthened the CaMbinding required for presentation of T286 as a substrate. In otherembodiments, compositions, for example, peptides comprising a portion ofthe CaM-KIIN molecule can be fused to a transporter agent. In certainembodiments, Tat-fused CN21 can be generated to increase cellpenetrating capabilities. In addition, compositions disclosed herein maybe used for studying cellular CaMKII function.

In certain embodiments, compositions and methods including a naturalCaMKII inhibitor protein CaM-KIIN can be used to inhibit CaMKII withlittle or no effect on CaMKI, CaMKIV, PKA or PKC. In other embodiments,two forms of CaM-KIIN are contemplated (e.g. isoforms). These isoformsare highly homologous to each other and co-localize with microtubules inneurons; both bind selectively to CaMKII only in its activated states.In more particular embodiments, compositions, methods and uses for shortCaM-KIIN-derived peptides are contemplated herein.

In certain embodiments, inhibitors of CaMKII are generated from variousregions of CaMKIIN inhibitory region. In one embodiment, minimalinhibitory CN regions are identified. In accordance with theseembodiments, kinase assays can be performed in order to assess certaininhibition of CaMK molecules such as CaMKII or CaMKI. In one example, amolecule referred to herein as CN19 is identified as a particularinhibitory region of CaM-KIIN having high potency (CN19, SEQ ID NO:2).Further truncations led to significant reduction in potency ofinhibition of CaMKII activity towards peptide substrates. (Note, ashorter core sequence is still able to significantly reducephosphorylation of protein substrates). In certain embodiments, potencyof CaMKII inhibition by CN19 can be modulated by mutations. One of amore notable increase of potency was by the R14A mutations for example.Also notable, S12 can be mutated without detriment or even with slightbenefit to potency (only by very specific substitution, V and R, but notby others); this can prevent phosphorylation of S12 and thereby preventinactivation (see FIG. 22B).

In certain embodiments, increased inhibition potency has been observedfor other CN19 molecules disclosed herein. For example, other CN19molecules of use for selective targeting of CaMKII can include CN19o(SEQ ID NO: 24), CN19a2v (SEQ ID NO: 23) or others. In accordance withthese embodiments, any inhibitor disclosed herein can be used as apharmaceutical composition (e.g. in combination with a pharmaceuticallyacceptable excipient) and administered to a subject to selectivelyinhibit target molecules or interrupt or promote regulatory cascadescontemplated herein. In certain embodiments, target molecules can beCaMKI and/or CaMKII or selectively inhibiting CaMKII in a subject inneed thereof. In other embodiments, any molecule disclosed herein can beused as a composition for administration to a subject in need thereofSome embodiments contemplated herein are directed at using CN19o (SEQ IDNO: 24) in a pharmaceutically acceptable composition and administered toa subject in need thereof. Depending on the need as determined by ahealthcare provider, compositions disclosed herein may be used to weaklyinhibit activity of a target molecule or to strongly inhibit orcompletely shut down the activity of a target molecule in a subject inneed thereof. It is contemplated herein that administration of acomposition disclosed herein can be used to temporarily affect activityof a target molecule or for longer term application which can includecomplete inhibition of activity of a target molecule.

In certain embodiments, fusion of truncated forms or portions of CaMKIINto cell penetrating agents can be performed. Cell penetrating agentscontemplated herein can include, but is not limited to, tat, ant,meristyl-groups, palmityl-groups, and other related or derived peptideand lipid compounds, as well as mimicking non-peptide and non-lipidcompounds. In certain examples, tat can be fused or covalently ornon-covalently attached to CN peptides, for example fused to CN21, CN19,CN19 variants (e.g., CN19o), CN17 or CN27 or mutant or variant thereofDifferent forms of fusion can be performed in order to design certainmolecules. For example, fusions may be at the C-terminus instead of theN-terminus, use different cell penetration-mediating compound; additionor reduction of linker sequences; use of overlapping sequences; fusionby other covalent or non-covalent means, including by disulfide bondsbetween added cysteines, which would be cleaved within cells.

In some embodiments, CN19 was demonstrated as maximal truncation notresulting in significant reduction of inhibitory potency althoughfurther truncations still show significant inhibition of CaMKIIactivity. In other embodiments, a 14 mer derived from CaMKIINα was stilleffective for inhibition of protein but not peptide phosphorylation byCaMKII. In addition, CN19 also retained specificity (e.g. it did notsignificantly affect the closely related protein kinase CaMKI).

Other embodiments herein relate to obtaining portions of CaM-KIIN andmutating or substituting one or more amino acids in the portions toalter potency of the portions for inhibiting CaMKII activity. In certainembodiments, mutations are generated to increase CaMKII inhibition. Forexample, mutational analysis of CN19 demonstrated that inhibitorypotency can be significantly further enhanced. In one example, anAlanine (Ala) scan (individual amino acid substitutions with Ala)identified 3 amino acid positions (P3, K13, R14) for which alaninesubstitutions/mutations clearly enhanced inhibitory potency. Severaladditional amino acid positions had slightly enhanced potency or noeffect. Additionally, amino acid positions can be replaced with positiveresidues (R or K) without loss of inhibitory potency. For example, thiscould be used to further enhance cell-penetration. In other embodiments,it is contemplated that molecules of CaM-KIIN portions can be altered toreduce phosphorylation of the portions which can alter potency ofinhibition. For example, CN19 contains one amino acid, Ser12, that couldbe phosphorylated (e.g. by PKC) within cells, thereby reducing CN19'sinhibitory effect. In one example, a single mutant R14A was very potent.S12 can be mutated, for example to R or V, without loss of potency. Incertain methods disclosed herein, it may be desirable to have acomposition having increased inhibitory potency compared to a wild typeCN molecule composition. In other embodiments, it was demonstrated thatCN19o potency was improved by over 100 fold with an IC50 of less than 1nM. In addition, CN19o (SEQ ID NO: 24) demonstrated about 50,000 foldselectivity of inhibition of CaMKII over CaMKI.

In some embodiments, it may be desirable to regulate the inhibition ofCaMKII by any peptide disclosed herein whether in a subject or in alaboratory setting. In accordance with embodiments, post-translationalmodifications or other cellular signaling methods may be used toregulate the peptide's activities regarding inhibition of CaMKII orCaMKI. For example, a protein kinase can be used to phosphorylate one ormore peptides disclosed herein to reduce the inhibition potency of thepeptide (e.g. S12 of CN19o can be phosphorylated), thus providingmethods for closely regulating CaMKII and CaMKIINα activities.

In certain embodiments, CN inhibitors contemplated herein can be made tobe cell-penetrating. In other embodiments, compositions contemplatedherein may be administered to a cell or to a subject by any method knownin the art (e.g. microbeads, microspheres, microparticles, slow releasegel). In other embodiments, CN inhibitors can be made for penetrating acell by for example, covalent fusion with the cell-penetrating ant(derived from the antennapedia protein; also called penetratin, SEQ IDNO:16) or tat (from the HIV tat protein, SEQ ID NO:15) sequences.However, in certain exemplary methods ant was found to interfere withgeneral calmodulin signaling (thereby interfering with specificity) bydirect binding to calmodulin, and effect further enhanced by ant fusionto CN27 (SEQ ID NO:17). By contrast, tat or tatCN21 did not bind tocalmodulin, making tatCN peptides viable cell penetrating inhibitors.Any compositions and methods known in the art for generatingcell-penetrating CN inhibitors or modulators is contemplated herein.

Other alternative methods may be used to make CN peptides cellpenetrating including, but not limited to, fusion with other cellpenetrating peptide sequences (e.g. various modifications of the tatsequence) or lipophilic compounds (such as myristyl groups). Thesefusions can be either at the N-terminus or at the C-terminus, with orwithout additional linkers or with removal of partially overlapping orexchangeable sequences. Any such fusions can also be made throughdisulfide bonds, allowing cleavage of the CN compound and thecell-penetration mediating compound in the reducing environment withincells, or other non-covalent or covalent bonds.

In certain embodiments, cell-penetrating CN inhibitors can protect fromglutamate excitotoxicity. In other embodiments, cell-penetrating CNinhibitors can protect from glutamate excitotoxicity after insult.tatCN21 showed a significant neuro-protective effect during glutamateexcitotoxicity, a cell culture model of stroke and other neurologicalconditions. Importantly, neuro-protection was observed even when thecompound was applied significantly after the insult, thus opening aclinically relevant window of therapeutic opportunity. In one example,tatCN21 showed significant neuroprotection both when present duringinsult and when added after insult. Some embodiments concernidentification of a drug target for post-insult neuro-protection.Knowledge of the actual drug targets will allow a relatively easyhigh-throughput screening for additional and/or alternative therapeuticcompounds. It is contemplated herein that CaMKII can be targeted usingmolecules and constructs disclosed and that new constructs and moleculesof interest can be identified that selectively inhibit CaMK molecules,for example CaMKII.

Neuronal Cell Death

In certain embodiments, glutamate excitotoxicity is contemplated to be acause of neuronal cell death in acute conditions. Conditions include,but are not limited to stroke, global ischemia (e.g. caused bysuffocation or cardiac arrest), or traumatic brain injury (e.g. causedby accidents, assaults, and explosions). Additionally, glutamate isconsidered to be involved in chronic neurodegenerative diseases. Chronicneurodegenerative diseases include, but are not limited to Alzheimer'sand Parkinson's. It is contemplated that compositions disclosed hereincan be of use to reduce, prevent or treat conditions that cause neuronaldeath in a subject in need thereof In other embodiments, compositionsand methods disclosed herein may be of use as novel inhibitors forsubjects having an addiction or undergoing cancer treatment. Forexample, CaMKII has been demonstrated to be involved in addiction, andCaMKII inhibitors have been shown to promote death of cancer cells (andto enhance the effect of other cancer-cell treating drugs). Therefore,it is contemplated herein that compositions and methods herein may beused to treat a subject having cancer. In certain embodiments,compositions and methods disclosed herein may be combined with otheranti-cancer treatments (e.g. radiation, chemotherapy, hyperthermia) fortreating a subject having cancer. In accordance with these embodiments,it is contemplated that CN21 and/or CN19 conserved, or with one or moremutation or addition, may be of use to treat a subject having cancer. Incertain embodiments, compositions and methods disclosed herein may be ofuse to promote cancer cell death in a subject.

Some embodiments herein concern administering a therapeuticallyeffective amount of a composition disclosed herein to a subject having aneurodegenerative disorder. In certain embodiments, compositionsdisclosed herein may be of use to reduce, prevent inhibit and/or treatconditions leading to neuronal death in a subject in need thereof Inother embodiments, compositions and methods disclosed herein may becombined with any treatment for neurodegenerative disorders orconditions causing neuronal cell death known in the art.

Nucleic Acids

As described herein, an aspect of the present disclosure concernsisolated nucleic acids and methods of use of isolated nucleic acids. Theterm “nucleic acid” is intended to include DNA and RNA and can be eitherbe double-stranded or single-stranded. In a preferred embodiment, thenucleic acid is a cDNA comprising a nucleotide sequence such as found inGenBank. In certain embodiments, the nucleic acid sequences disclosedherein have utility as hybridization probes or amplification primers.These nucleic acids may be used, for example, in diagnostic evaluationof tissue samples. In certain embodiments, these probes and primersconsist of oligonucleotide fragments. Such fragments should be ofsufficient length to provide specific hybridization to a RNA or DNAtissue sample. The sequences typically will be 10-20 nucleotides, butmay be longer. Longer sequences greater than 50 even up to full length,are preferred for certain embodiments.

In certain embodiments, it will be advantageous to employ nucleic acidsequences in combination with an appropriate means, such as a label, fordetermining hybridization. A wide variety of appropriate indicator meansare available (i.e. fluorescent, radioactive, enzymatic or otherligands, such as avidin/biotin) that are capable of being detected. Inpreferred embodiments, one may desire to employ a fluorescent label oran enzyme tag such as urease, alkaline phosphatase or peroxidase,instead of radioactive or other environmentally undesirable reagents. Inthe case of enzyme tags, colorimetric indicator substrates are knownwhich can be employed to provide a detection means visible to the humaneye or spectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will not only be useful in solutions as in PCR, for detection ofexpression of corresponding genes but also in embodiments employing asolid phase. In embodiments involving a solid phase, the test DNA (orRNA) is adsorbed or otherwise affixed to a selected matrix or surface.This fixed, single-stranded nucleic acid is then subjected tohybridization with selected probes under known conditions.

The gene or gene fragment encoding a polypeptide (e.g. CaM-KIIN orportion thereof) may be inserted into an expression vector by standardsubcloning techniques. An E. coli expression vector may be used whichproduces the recombinant polypeptide as a fusion protein, allowing rapidaffinity purification of the protein. Examples of such fusion proteinexpression systems are the FLAG system (IBI, New Haven, Conn.), and the6.times.His system (Qiagen, Chatsworth, Calif.).

Any expression vector (e.g. mammalian, yeast, bacterial etc.) known inthe art is contemplated for expression of CaM-KIIN constructs orderivatives, or other CaMKII inhibitor molecule constructs etc.

A recombinant expression vector may be a plasmid. The recombinantexpression vector further may be a virus, or portion thereof, whichallows for expression of a nucleic acid introduced into the viralnucleic acid.

Recombinant expression vectors can be designed for expression ofpeptides or peptide constructs contemplated herein. For example,proteins can be expressed in bacterial cells such as E. coli, insectcells (using baculovirus), yeast cells or mammalian cells.

One embodiment includes isolated nucleic acids encoding proteins havingbiological activity of CaMKII inhibition. The term “isolated” refers toa nucleic acid substantially free of cellular material or culture mediumwhen produced by recombinant DNA techniques, or chemical precursors orother chemicals when chemically synthesized. An “isolated” nucleic acidis also free of sequences that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in theorganism from which the nucleic acid is derived.

It will be appreciated that isolated nucleic acids includes nucleicacids having substantial sequence homology with the nucleotide sequenceof portions of CaM-KIIN that have CaMKII inhibitory activity.

Proteins comprising an amino acid sequence that is 50%, 60%, 70%, 80% or90% homologous with the amino acid of CaM-KIIN may provide proteinshaving CaMKII inhibitory activity.

A nucleic acid of the embodiments, for instance an oligonucleotide, canalso be chemically synthesized using standard techniques. Variousmethods of chemically synthesizing polydeoxynucleotides are known,including solid-phase synthesis which, like peptide synthesis, has beenfully automated in commercially available DNA synthesizers (See i.e.,Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No.4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

Protein Purification

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or analysis by SDS/PAGE toidentify the number of polypeptides in a given fraction. A preferredmethod for assessing the purity of a fraction is to calculate thespecific activity of the fraction, to compare it to the specificactivity of the initial extract, and to thus calculate the degree ofpurity, herein assessed by a “-fold purification number”. The actualunits used to represent the amount of activity will be dependent uponthe particular assay technique chosen to follow the purification andwhether or not the expressed protein or peptide exhibits a detectableactivity.

Methods for purifying various forms of proteins are known. (i.e.,Protein Purification, ed. Scopes, Springer-Verlag, New York, N.Y., 1987;Methods in Molecular Biology: Protein Purification Protocols, Vol. 59,ed. Doonan, Humana Press, Totowa, N.J., 1996). The methods disclosed inthe cited references are exemplary only and any variation known in theart may be used. Where a protein is to be purified, various techniquesmay be combined, including but not limited to cell fractionation, columnchromatography (e.g., size exclusion, ion exchange, reverse phase,affinity, etc.), Fast Performance Liquid Chromatography (FPLC), HighPerformance Liquid Chromatography (HPLC), gel electrophoresis,precipitation with salts, pH, organic solvents or antibodies,ultrafiltration and/or ultracentrifugation.

There is no general requirement that the protein or peptide always beprovided in the most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. Methods exhibiting a lower degreeof relative purification may have advantages in total recovery ofprotein product, or in maintaining the activity of an expressed protein.

One embodiment provides isolated proteins having biological activity ofCaMKII inhibition. In a certain embodiments, a protein having biologicalactivity of CaMKII inhibition comprises an amino acid sequence found inCaM-KIIN. In other embodiments, a protein having biological activity ofCaMKII inhibition comprises an amino acid sequence, aptamer, antibody orantibody fragment or small molecule that is capable of reducing oreliminating CaMKII activity. Other proteins having biological activityof CaMKII inhibition may have substantial sequence homology to the aminoacid sequence of CaM-KIIN, and are also encompassed herein. Contemplatedherein are use of these proteins, aptamers, antibody, antibody fragmentand/or small molecules with CAMKII inhibitor activity to treat a subjectincluding, but not limited to, a subject experiencing neuronal celldeath, having had an acute insult (e.g. stroke, global cerebralischemia, traumatic brain injury), having a drug addiction, havingcancer, or having a neurodegenerative disorder.

In certain embodiments, compositions disclosed herein, for example,compositions having a peptide can include a derivative of the peptidewherein one or more amino acids can be substituted with one or morearginine, alanine, valine, lysine or combination thereof or other aminoacid substitutions or mutations thereof (e.g. wherein the amino acidsequence is 19 or more consecutive amino acids of SEQ ID NO:1, 2 or 17).

Molecules which bind to a protein including the antibodies, bispecificantibodies and tetrameric antibody complexes, can be used in a methodfor identifying CaMKII inhibitory molecules by labeling a molecule witha detectable substance, contacting the molecule with cells and detectingthe detectable substance bound to the cells.

Another method for the preparation of the polypeptides may use peptidemimetics. Mimetics are peptide-containing molecules that mimic elementsof protein secondary structure. The underlying rationale behind the useof peptide mimetics is that the peptide backbone of proteins existschiefly to orient amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is expected to permit molecular interactions similar to thenatural molecule (e.g. CaMKII inhibitory activity). An embodimentincludes the use of protein mimetics to mimic the CaMKII inhibitoryactivity of constructs and molecules disclosed herein.

Carriers (Lipids, Liposomes, Micelles, Polymers, and Nanoparticles)

Any methods for formation of liposomes and micelles may be use and areknown in the art. Nanoparticles or nanocapsules formed from polymers,silica, or metals, which are contemplated herein for drug delivery orimaging, have been described. It is contemplated that delivery ofconstructs disclosed herein may carried out using the above referencedcarriers or any carrier known in the art.

Imaging Agents and Radioisotopes

In certain embodiments, molecules, for example, peptides or proteins maybe attached to imaging agents of use for imaging and diagnosis ofvarious diseased organs, tissues or cell types. Many appropriate imagingagents are known in the art, as are methods for their attachment toproteins or peptides. Certain attachment methods can involve the use ofa metal chelate complex employing, for example, an organic chelatingagent such a DTPA attached to the protein or peptide. Target moleculesalso may be reacted with an enzyme in the presence of a coupling agentsuch as glutaraldehyde or periodate. Conjugates with fluorescein markersare prepared in the presence of these coupling agents or by reactionwith an isothiocyanate.

Non-limiting examples of paramagnetic ions of potential use as imagingagents include chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III), with gadolinium beingparticularly preferred. Ions useful in other contexts, such as X-rayimaging, include but are not limited to lanthanum (III), gold (III),lead (II), and especially bismuth (III).

Radioisotopes of potential use as imaging or therapeutic agents includeastatine²¹¹, ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt,copper⁶², copper⁶⁴ , copper⁶⁷, ¹⁵²Eu, fluorine¹⁸, gallium⁶⁷, gallium⁶⁸,³hydrogen, iodine¹²³, iodine¹²⁴, iodine¹²⁵, iodine¹³¹, indium¹¹¹,⁵²iron, ⁵⁹iron, ³²phosphorus, ³³phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸,Sc⁴⁷, ⁷⁵selenium, silver¹¹¹, ³⁵sulphur, technicium^(94m)technicium^(99m) yttrium⁸⁶ and yttrium⁹⁰. ¹²⁵I is often being preferredfor use in certain embodiments, and technicium^(99m) and indium¹¹¹ arealso often preferred due to their low energy and suitability for longrange detection.

Radioactively labeled proteins or peptides may be produced according towell-known methods in the art. For instance, they can be iodinated bycontact with sodium or potassium iodide and a chemical oxidizing agentsuch as sodium hypochlorite, or an enzymatic oxidizing agent, such aslactoperoxidase. Proteins or peptides may be labeled withtechnetium-^(99m) m by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the peptide to this column or bydirect labeling techniques, e.g., by incubating pertechnate, a reducingagent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the peptide. Intermediary functional groupswhich are often used to bind radioisotopes which exist as metallic ionsto peptides include diethylenetriaminepentaacetic acid (DTPA), DOTA,NOTA, porphyrin chelators and ethylene diaminetetracetic acid (EDTA).Also contemplated for use are fluorescent labels, including rhodamine,fluorescein isothiocyanate and renographin.

In certain embodiments, the claimed peptides or constructs may be linkedto a secondary binding ligand or to an enzyme (an enzyme tag) that willgenerate a colored product upon contact with a chromogenic substrate.Examples of suitable enzymes include urease, alkaline phosphatase,(horseradish) hydrogen peroxidase and glucose oxidase. Preferredsecondary binding ligands are biotin and avidin or streptavidincompounds. The use of such labels is well known to those of skill in theart. These fluorescent labels are preferred for in vitro uses, but mayalso be of utility in vivo applications, particularly endoscopic orintravascular detection procedures.

In alternative embodiments, molecules herein may be tagged with afluorescent marker. Non-limiting examples of photodetectable labelsinclude Alexa 350, Alexa 430, AMCA, aminoacridine, BODIPY 630/650,BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX,5-carboxy-4′,5′-dichloro-2′,7′-dimethoxy fluorescein,5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein,5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino,Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansyl chloride, Fluorescein, HEX,6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole), Oregon Green 488, OregonGreen 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalicacid, isophthalic acid, cresyl fast violet, cresyl blue violet,brilliant cresyl blue, para-aminobenzoic acid, erythrosine,phthalocyanines, azomethines, cyanines, xanthines, succinylfluoresceins,rare earth metal cryptates, europium trisbipyridine diamine, a europiumcryptate or chelate, diamine, dicyanins, La Jolla blue dye,allopycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine,phycoerythrocyanin, phycoerythrin R, REG, Rhodamine Green, rhodamineisothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), Tetramethylrhodamine, and Texas Red. These andother luminescent labels may be obtained from commercial sources such asMolecular Probes (Eugene, Oreg.).

Chemiluminescent labeling compounds of use may include luminol,isoluminol, an aromatic acridinium ester, an imidazole, an acridiniumsalt and an oxalate ester, or a bioluminescent compound such asluciferin, luciferase and aequorin. Diagnostic immunoconjugates may beused, for example, in intraoperative, endoscopic, or intravascular tumoror disease diagnosis.

In various embodiments, labels of use may comprise metal nanoparticles.Methods of preparing nanoparticles are known. Nanoparticles may also beobtained from commercial sources (e.g., Nanoprobes Inc., Yaphank, N.Y.).Modified nanoparticles are available commercially, such as Nanogold®nanoparticles from Nanoprobes, Inc. (Yaphank, N.Y.). Functionalizednanoparticles of use for conjugation to proteins or peptides may becommercially obtained.

Site-Specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art. As will be appreciated, the technique typically employs abacteriophage vector that exists in both a single stranded and doublestranded form. Typical vectors useful in site-directed mutagenesisinclude vectors such as the M13 phage. These phage vectors arecommercially available and their use is generally well known to thoseskilled in the art. Double stranded plasmids are also routinely employedin site directed mutagenesis, which eliminates the step of transferringthe gene of interest from a phage to a plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, and subjected to DNA polymerizingenzymes such as E. coli polymerase I Klenow fragment, in order tocomplete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants. Any technique known in the art for generating one or moreamino acid changes or mutations in constructs disclosed herein iscontemplated (e.g. CaM-KIN derived constructs).

Pharmaceutical Compositions and Routes of Administration

Aqueous compositions contemplated herein may include an effective amountof a therapeutic peptide, peptide construct, epitopic core region,stimulator, inhibitor, and the like, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Aqueouscompositions of gene therapy vectors expressing any of the foregoing arealso contemplated. The phrases “pharmaceutically or pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic or other untoward reaction whenadministered to an animal, or a human, as appropriate.

Aqueous compositions contemplated herein may include an effective amountof the compound, dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Such compositions can also be referred to asinocula. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions. For human administration,preparations should meet sterility, pyrogenicity, general safety andpurity standards as required by FDA Office of Biologics standards.

Biological material should be extensively dialyzed to remove undesiredsmall molecular weight molecules and/or lyophilized for more readyformulation into a desired vehicle, where appropriate. The activecompounds will then generally be formulated for parenteraladministration, e.g., formulated for injection via the intravenous,intramuscular, sub-cutaneous, intralesional, or even intraperitonealroutes. The preparation of an aqueous composition that contains anactive component or ingredient will be known to those of skill in theart in light of the present disclosure. Typically, such compositions canbe prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for use in preparing solutions or suspensions uponthe addition of a liquid prior to injection can also be prepared; andthe preparations can also be emulsified.

Pharmaceutical forms suitable for injectable use include sterile aqueoussolutions or dispersions; formulations including for example, aqueouspropylene glycol; and sterile powders for the extemporaneous preparationof sterile injectable solutions or dispersions. In all cases the formmust be sterile and must be fluid. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

A therapeutic agent can be formulated into a composition in a neutral orsalt form. Pharmaceutically acceptable salts, include the acid additionsalts (formed with the free amino groups of the protein) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. In terms of using peptide therapeutics as active ingredients, anymethod known in the art may be used.

Carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The preparation of more, or highly, concentratedsolutions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion.

The term “unit dose” refers to physically discrete units suitable foruse in a subject, each unit containing a predetermined quantity of thetherapeutic composition calculated to produce the desired responses,discussed above, in association with its administration, i.e., theappropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the subject to be treated, the state of the subject and theprotection desired. The person responsible for administration will, inany event, determine the appropriate dose for the individual subject.

The active therapeutic agents may be formulated within a mixture tocomprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose orso. Multiple doses can also be administered, daily, weekly, bi-weekly ormonthly for example.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g., tablets or other solids for oraladministration; liposomal formulations; time release capsules; and anyother form currently used.

One may also use nasal solutions or sprays, aerosols or inhalants in thepresent invention. Nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions. Thus, the aqueous nasal solutions usually areisotonic and slightly buffered to maintain a pH of 5.5 to 6.5. Inaddition, antimicrobial preservatives, similar to those used inophthalmic preparations, and appropriate drug stabilizers, if required,may be included in the formulation.

Additional formulations which are suitable for other modes ofadministration include suppositories and pessaries. A rectal pessary orsuppository may also be used. Suppositories are solid dosage forms ofvarious weights and shapes, usually medicated, for insertion into therectum or the urethra. After insertion, suppositories soften, melt ordissolve in the cavity fluids. In general, for suppositories,traditional binders and carriers may include, for example, polyalkyleneglycols or triglycerides; such suppositories may be formed from mixturescontaining the active ingredient in the range of 0.5% to 10%, preferably1% 2%.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders. Incertain defined embodiments, oral pharmaceutical compositions willcomprise an inert diluent or assimilable edible carrier, or they may beenclosed in hard or soft shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 75% of theweight of the unit, or preferably between 25-60%. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring.

In one embodiment, doses may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 5 or more years. Persons ofordinary skill in the art may estimate repetition rates for dosing basedon measured residence times and concentrations of the targetableconstruct or complex in bodily fluids or tissues. Following successfultreatment, it may be desirable to have the patient undergo maintenancetherapy to prevent the recurrence of the disease state, wherein thetherapeutic agent is administered in maintenance doses, ranging from0.01 ug to 100 mg per kg of body weight, once or more daily, to onceevery 5 years.

In another embodiment, a particular dose may be calculated according tothe approximate body weight or surface area of the patient. Otherfactors in determining the appropriate dosage can include the disease orcondition to be treated or prevented, the severity of the disease, theroute of administration, and the age, sex and medical condition of thepatient. Further refinement of the calculations necessary to determinethe appropriate dosage for treatment is routinely made by those skilledin the art, especially in light of the dosage information and assaysdisclosed herein. The dosage can also be determined through the use ofknown assays for determining dosages used in conjunction withappropriate dose-response data.

Kits

Some embodiments concern kits for compositions and methods disclosedherein. A kit can include, but is not limited to, one or morecompositions in one or more containers or vessels for reducing,inhibiting, treating and/or preventing CaMKII activity (e.g. forreducing neuronal cell death or inhibiting a neurodegenerativedisorder). Alternatively, a kit may include only reagents, CaMKIINαderived molecules or constructs for further research of potency oninhibiting CaMKII activity or other activity. In addition, kits arecontemplated of use for health professionals in treating disorders andconditions indicated herein.

In yet other embodiments, a kit can include one or more peptides derivedfrom CaMKIINα (e.g. CN21, CN27, CN19 etc.). Kits contemplated of useherein can include a kit for medical applications or kits for researchapplications for further study.

EXAMPLES

Embodiments herein are further illustrated by the following examples anddetailed protocols. However, the examples are merely intended toillustrate embodiments and are not to be construed to limit the scopeherein. The contents of all references and published patents and patentapplications cited throughout this application are hereby incorporatedby reference.

Example 1

In certain exemplary methods, portions or peptides of CaM-KIIN aregenerated. In certain examples, peptides are 21 amino acids in length.As represented in FIG. 1. CN21a (CN) contains full CaMKII inhibitorypotency and specificity of CaM-KIINtide. FIG. 1A represents CN peptidesrelative to the sequence of CaM-KIINtide, termed CN27. Darker barsindicate greater CaMKII inhibitory potential found in this study. InFIG. 1B, CN21a contains the full inhibitory potency. Deletion of the 3N-terminal amino acids of CN21a abolishes inhibition, while C-terminaldeletion reduces it. In another example, Ca²⁺/CaM-induced CaMKIIactivity was measured by ³²P incorporation into the peptide substrateAC2. In FIG. 1C, CN21a specificity was tested on a panel of differentkinases (at 5 μM, 50-fold IC50). Kinase activity without inhibitor wasnormalized to 100% for each kinase. CaMKII activity was completelyblocked, while activities of the other kinases were not affected.Individual data points of duplicate assays are shown. In FIG. 1D, CN27and CN21 were demonstrated to inhibit both major brain CaMKII isoforms,α and β. Error bars indicate s.e.m.

In another exemplary experiment, CNs were demonstrated to differentiallyinhibit phosphorylation of two peptide substrates (see FIGS. 2A-2C). Inone example, FIG. 2A represents that CN peptides (e.g. 1 μM) blockCaMKII phosphorylation of the peptide substrate syntide2 (derived from aphosphorylation site on glycogen synthase), but only reducephosphorylation of AC2 (derived from the auto-phosphorylation sitearound T286). FIG. 2B represents that AC2 interferes with CaMKIIinhibition by CN21. Kinase assays were done in presence of 0.1 or 0.5 μMCN21 with 80 μM syntide2 as the principle substrate; AC2 was added atthe indicated concentration. Kinase activity is shown as % of maximalactivity without CN21 peptide. Error bars indicate s e m Inhibition byCN21 is competitive with AC2, demonstrated in FIG. 2C. Standard kinaseassays were performed with 10 nM CaMKII, 1 μM CaM, and varyingconcentrations of AC2 (40, 13.33, 8, and 5 μM) and CN21 (0, 0.3, 0.6,and 0.9 μM). In a Lineweaver-Burk plot, increasing the CN21concentration has a much stronger effect on apparent −1/k_(m) (x-axisintersection) than on apparent 1/ν_(max) (y-axis intersection),indicating inhibition by a largely competitive mechanism. The r² valuesof regression for all inhibitor series were >0.994.

In another example, FIG. 3. CaM-KIIN and CN21 have been demonstrated tointeract with the CaMKII T-site. FIG. 3A represents a model of CaMKIIkinase domain (Rosenberg et al., 2005) with the substrate binding S-site(indicated), the T286-region binding T-site (indicated), and theregulatory region (shown as ribbon). The arrow indicates the proposedorientation of CN21a binding to the T-site, when the regulatory regionis displaced after activation. FIG. 3B represents a comparison toGFP-CaMKIIα wild type (wt), the activity of the CaMKII T-site mutantsW237R and 1205K were significantly less affected by CN21a (p<0.001; n=6)or CaM-KIIN (p<0.02; n=4), as assessed by standard kinase assays withthe peptide substrate syntide2. Results are normalized to maximal kinaseactivity without inhibitor. Error bars show s.e.m. FIG. 3C represents anillustration demonstrating that CN21 efficiently blockedCa²⁻/CaM-induced CaMKII binding to immobilized GST-NR2B-c, aninteraction that occurs at the CaMKII T-site. Bound CaMKII was elutedand detected by Western blot. C- but not N-terminal truncations of CN21also inhibited binding to NR2B.

FIG. 4 illustrates an experiment demonstrating that CaM-KIIN and CN21a(5 μM) slowed down CaM dissociation from CaMKII (150 nM). Dissociationof TA-CaM (30 nM) was monitored by its increased fluorescence (1 secsample times) during a chase with excess unlabeled CaM (60 μM). Thecontrol peptide CN21c did not slow down dissociation. CN21a sloweddissociation from CaMKII, but not from a peptide derived from the CaMKIICaM-binding domain (CBD), demonstrating a CaMKII directed effect.

Other experiments were performed to assess whether CaMKIIautophosporylation was blocked by CaM-KIIN or CN21. FIGS. 5A-5Cillustrate exemplary gels that CaM-KIIN and CN21 block substrate- andT305 but not T286 auto-phosphorylation. FIG. 5A represents CN peptides(5 μM) blocked CaMKII substrate- but not T286 auto-phosphorylation whenstimulated with 1 μM CaM (5 min reaction time). Phosphorylation of MAP2and CaMKII autophosphorylation at T286 were assessed byWestern-analysis. Only CN21c failed to block MAP2 phosphorylation,indicating CN21a amino acids 4-14 as core inhibitory region.Importantly, none of the CN peptides blocked CaMKII T286auto-phosphorylation. FIG. 5B represents a time course of CaMKIIauto-phosphorylation stimulated by 0.1 μM CaM at 30° C. CN21 and KN93 (5and 10 μM, respectively) blocked T305 and other auto-phosphorylationthat result in a band-shift of CaMKII. By contrast, T286 was essentiallycompletely blocked by KN93, but not by CN21. Total CaMKII andauto-phosphorylation at T286 and T305 were detected by Western-analysis.FIG. 5C represents a slot-blot analysis (left panel) was performed forquantification (right panel) of T286-auto-phosphorylation in theexperiment shown in B. T286 auto-phosphorylation was normalized to thedegree seen after 8 min reaction without inhibitor; dilutions of thisreaction were used as standard. Slot-blot avoids differences in the areaof signal cause by the band-shift seen only in absence of inhibitor.CN21 had no significant effect on the T286-auto-phosphorylation measured(p>0.25). Error bars show s.e.m. of triplicates. FIG. 5D representswhere reactions as in B (submaximal CaM), were slowed down further bylow temperature (on ice). Under these conditions, CN inhibitors sloweddown T286 auto-phosphorylation, but still did not completely block it.T305 and other auto-phosphorylation that result in band shift were notdetected under these conditions.

In another exemplary method, a construct was made where a portion of aCaMKIINα molecule, CN21 was made more cell penetrating. This molecule isreferred to as tatCN21. tatCN21 was demonstrated to retain inhibition ofCaMKII activity and binding to NR2B. FIG. 6A illustrates the effect oftatCN21 on a panel of different kinases. Kinase activity withoutinhibitor was normalized to 100% for each kinase. 1 μM tatCN21completely blocked CaMKII activity, while even 5 μM tatCN21 had littleor no effect on the other kinases: a mild but clear effect was observedonly on CaMKIV (˜35%). Individual data points of duplicate assays areshown. FIG. 6B illustrates that similar to CN21a, both tatCN21 andantCN27 efficiently blocked Ca²⁻/CaM-induced CaMKII binding toimmobilized GST-NR2B-c. Control peptides did not affect CaMKII binding.Bound CaMKII was eluted and detected by Western blot.

Example 2

In another example, motility of hippocampal neurons was analyzed. Asillustrated in FIG. 7, tatCN21 inhibits motility in hippocampal neurons.Images of GFP expressing hippocampal neurons (5-6 days in vitro) wereacquired at 30° C. in 20 s intervals in order to assess motility. Afterthe first set of 16 images, neurons were incubated with tatCN21a or withcontrol peptide for 20 min; then a second set of images was taken. Scalebars: 10 μm. FIG. 7A represents exemplary images of the same dendritearea before and 20 min after addition of tatCN21, at different times ofacquisition as indicated. A images were created by subtracting pixelintensities of one image from the one taken 20 s later. Shown A imageintensities are 4 fold exaggerated compared to the original captures onthe left. FIG. 7B represents an average of A images in pseudo colorvisualize motility in a larger area (110×148 μm). Error indicates s.e.m.of the A images used for the average projections shown. FIG. 7Crepresents quantification changes in motility after application oftatCN21 or tatRev control based on A image average projections. Errorbars show s.e.m. (n=7 neurons from three independent cultures). In threecases, neurons were treated first with tatRev and then tatCN21, as shownin B.

In another exemplary method, a portion of a CaM-KIIN molecule wasexamined for effects on glucose induced insulin secretion. FIG. 8represents that tatCN21 inhibits glucose-induced insulin secretion.Insulin secretion from acutely isolated rat Langerhans' islets (10 perwell) was stimulated by 11 mM glucose. This secretion was inhibited byextracellular EGTA (0.5 mM; instead of 2.5 mM CaCl₂), KN93 (10 μM),antCN27 (5 μM) and tatCN21 (5 μM) (p<0.025; for KN93 and tatCN21p<0.01). Error bars show s.e.m. (n=4; for antCN27 n=3). The CN peptidesinhibited secretion also in an independent experiment (which did notinclude the standard for determining absolute insulin amount; notshown).

In certain exemplary methods, peptides and even proteins can be madecell-penetrating by fusion to the arginine/lysine-rich ant or tatsequences (FIG. 9A), providing powerful tools for studying cellularfunctions. A concern usually tested for is the possibility that fusionmay disrupt activity of the original compound. Here, we provide acautionary tale that any ant fusion can also generate new undesiredeffects by direct binding to calmodulin (CaM).

In another exemplary method, a fusion molecule was linked to CN. In thisexample, ant fusion was performed to CaMKIIN inhibitory region (CN; FIG.9A) in order to generate a potent cell-permeable CaMKII inhibitor,antCN27. Here, ant but not tat fusion with CN peptides generated anadditional CaM-directed mode of inhibition (FIG. 9B and 9C) thatcompromised specificity (FIG. 9D). The CaM-directed mode but not theCaMKII-specific mode of inhibition was also shared by the reversesequence control peptide antRev (FIG. 9B). Tat fusion to the newlyidentified minimal inhibitory region of CaM-KIIN, CN21, did not show theCaM-directed mode of inhibition (FIG. 9C) and largely retained thespecificity of non-fused CN21 (FIG. 9D). CN peptides efficiently blocksubstrate- and T305 but not T286 auto-phosphorylation of CaMKII(however, the autonomous activity generated by the T286auto-phosphorylation is blocked by CN peptides). The tat fusion peptidesshowed the same differential effect on substrate versus T286phosphorylation as non-fused CNs, whereas ant fusion peptidesadditionally inhibited T286 auto-phosphorylation by their CaM-directedmode (FIG. 10). Both ant- and tat-fused CNs blocked CaMKII binding toNR2B.

Direct binding of biotinylated CaM to ant immobilized by slot blotrevealed the mechanism of the CaM-directed mode of inhibition (FIG. 9E).Formally, the ant peptides competed with CaMKII for CaM binding. CaM didnot bind to the tat sequence or the non-fused CN peptides (FIG. 9E). Inan overlay assay, biotinylated CaM bound to calcineurin A (CaN-A),CaMKIIa, and other proteins from rat brain extract immobilized on PVDFmembranes (FIG. 9F). The ant fusion peptides interfered withCa¹⁺-dependent CaM binding, whereas tat fusion peptides and non-fused CNpeptides did not (FIG. 9F). The ant peptide alone also interfered withsuch CaM binding, but somewhat less efficiently than antCN27 (FIG. 9F).No significant interference with Ca²⁺-independent binding of CaM wasobserved. TA-labeled CaM represents reduced fluorescence after bindingto CaMKII or its CaM binding domain (CBD; −0.05 nM k_(D) for CaM⁵; seealso FIG. 9G). Surprisingly, binding to ant did not reduce TA-CaMfluorescence, preventing direct affinity measurement, but enablingcompetition experiments (FIG. 9G). Competition with CBD indicated ˜1 nMand ˜5 nM k_(D) of antCN27 and ant for binding to CaM, respectively,while tatCN21 did not show any competition (FIG. 9G).

Furthermore, motility was affected only by antCN27 but not antRev, whichshares the CaM-directed but not CaMKII-specific mode of inhibition (FIG.9B). Results show that ant binding to CaM can be enhanced even by fusionto peptides that do not detectably interact with CaM on their own (FIG.9E and 9G). Here, tat fusion provided a viable alternative for creatinga potent, specific, and cell-permeable CaMKII inhibitor, tatCN21 .

FIGS. 9A-9G illustrate that ant but not tat fusion compromisedspecificity of an inhibitor peptide by direct binding to calmodulin.(9A) The ant and tat sequences fused to the N-terminus of CNs; (K) wasreplaced by the first K of CNt in the fusion, (9B) Extent of CaMKIIinhibition by 1 u,M antCN27 and the reverse sequence control antRevdepended on the CaM concentration, indicating an additional CaM-directedmode of inhibition generated by ant fusion. Measured was AC2phosphorylation as described⁴. Error bars show s.e.m. (9C) 1 uM tatCN21strongly inhibited CaMKII-mediated AC3 phosphorylation at all CaMconcentrations, while the reverse and scrambled sequence controls tatRevand tatScr had no effect compared to assays without tat peptide. (9D)antCN27 blocked CaMKII activity, but also affected PKC and reducedCaMKIV activity by −70%. tatCN21 blocked CaMKII activity at both 1 and 5u,M, and reduced CaMKIV activity by −35% only at 5 U.M; other kinaseswere not affected. Kinase panel assays were done as described, (9E)Binding of biotinylated CaM (25 nM in TBS, pH 7.5, 1 mM CaCl₂) topeptides immobilized by slot blot, (9F) Interference of peptides (5 μM)with CaM binding (conditions as in e) to brain proteins in a blotoverlay assay. The major Ca²⁺-dependent CaM-binding proteins wereCalcineurin A (CaN-A) and CaMKIIoc; additional protein binding wasdetected after longer exposure (see FIGS. 11A-11B). AntCN27 (upperpanel) and ant (lower panel) affect Ca²⁺-dependent CaM binding, but notbinding to proteins also detected after Ca²⁺ was chelated by EGTA. (9G)antCN27 and ant, but not tatCN21, compete with the CaMKII CaM-bindingdomain (CBD) for binding of TA-labeled CaM. Fluorescence of TA-CaM isreduced by binding to CBD. Fluorescence (A_(,cx)=335 nm; X_(cm)=415 nm;1 s sample time) was monitored for 150 s after each addition of CBD aspreviously described.

FIG. 10 illustrates a differential effect of tat- and ant-CN fusionpeptides on CaMKII substrate- and auto-phosphorylation. Reactions wereperformed in presence of 1 uM CN peptides and 5 uM CaM (upper panels) or5 uM CN peptides and 1 uM CaM (lower panels), with 20 nM CaMKII and 10nM MAP2. Phosphorylation of MAP2 and CaMKII T286 auto-phosphorylationwere assessed by Western-analysis. tatCN21, but not the tat controlpeptide, blocked MAP2 but not T286 phosphorylation, as seen fornon-fused CN21. Similar results were obtained for the ant peptide pair,but only at low peptide/high CaM concentration. At high peptide/low CaM,both ant peptides interfered with autophosphorylation, as predictedbased on their additional CaM-competitive mode of inhibition generatedby ant fusion.

Example 3

In another example, neuronal cell death is measured by LDH release. Inthis exemplary method, glutamate was used to induce neuronal cell death,A portion of CaMKIIN was fused to a cell penetrating molecule togenerate tatCN21a construct. The construct was analyzed to examinewhether it was capable of attenuating neuronal cell death before andafter glutamate insult (see FIGS. 11A-11C). tatCN21a inhibited glutamateinduced neuronal cell death not only when present during the insult,but, also when added 1 h after the insult. By contrast, traditionalCaMKII inhibitor KN93 was protective only when present during theinsult, but not when added after the insult. This demonstrates that thenew CN inhibitors described here, but not the traditional KN inhibitors,are useful in a clinically relevant time window. Corresponding resultswere observed in dissociated cultures of rat hippocampal (FIG. 11A) andcortical (FIGS. 11B-11C) cultures, both after 7 days (FIGS. 11A-11B) andafter 18 days (FIG. 11C) in culture.

FIG. 11D represents an exemplary histogram illustrating that tatCN21inhibits glutamate induced cell death (7DIV cortical) when added 1-6hours post insult, but APV did not. APV, a NMDAR antagonist, onlyreduces cell death when present during the insult. Like KN93, it isineffective when added after insult.

FIG. 11E represents that siRNA targeting CaMKIIα (siα) reducesexpression of CaMKII in 10DIV hippocampal neurons as indicated bywestern-blot. Neurons were co-transfected with siRNA or non-targetingRNA (NT) at 8DIV and were assayed at day 10. Cells transfected at 8DIVwith 1-2 mg/well of sia in 24 well plates showed reduced CaMKIIa whenassayed at 10DIV by western-blot. Blots were re-probed for tubulin as aloading control. sia and sib reduce NMDA induced cell death in 10DIVhippocampal neurons. Cells were treated with 300 mM NMDA plus 50 mM CNQX(an AMPAR inhibitor) for 5 minutes and cell death was assayed 24 hoursafter insult. NMDA and CNQX were used because some evidence indicatesthat knockdown of CaMKII increases gluR1 expression levels and thusmight increase AMPAR expression. Increased AMPAR expression is thoughtto lead to increased cell death. Thus, by inducing cell death with NMDAand an AMPAR inhibitor, the AMPAR component was reduced. Also shown, ascontrol, tatCN21 reduces NMDA-induced cell death in 10DIV hippocampalneurons.

Example 4

FIGS. 12A-12D represent that KN93 and tatCN21 have different effects onCaMKII. FIG. 12A illustrates that both tatCN21 and KN93 inhibit calciumand calmodulin stimulated CaMKII activity, but only tatCN21 inhibitsautonomy. Stimulated CaMKII activity was assessed using purified CaMKII,[γ³²P]ATP, and the CaMKII substrate, syntide2. Reaction was initiated byaddition of calcium and calmodulin. To measure autonomous activity,CaMKII was pre-autophosphorylated by incubation with calcium andcalmodulin, which was then chelated by addition of EDTA prior to addingthe kinase to syntide2 and [γ³²P]ATP. FIG. 12B representsself-association in vitro is inhibited by both KN93 and tatCN21. Tomeasure self-association, purified CaMKII was incubated for five minuteswith 1 mM ADP, calcium and calmodulin, and the appropriate inhibitor.The reaction mixture was then centrifuged to pellet the self-associatedkinase. The supernatant (S) was removed and saved and the pellet (P) wasresuspended. Both were then resolved by SDS-PAGE and western-blotted forCaMKII. Self-associated kinase is found in the pellet. FIG. 12Crepresents binding to the immobilized NR2B subunit of the NMDAR in vitrowas determined in the presences or absence of tatCN21, KN93, or anon-specific kinase inhibitor, H7. TatCN21 and KN93 inhibited binding,but H7 did not. FIG. 12D represents a table summarizing the effects oftatCN21 and KN93 on CaMKII activity, NR2B binding, or self-association.

Example 5

FIG. 13A represents that overexpression of T286A reduces glutamateinduced cell death compared with overexpression of CaMKIIa. 7DIVhippocampal neurons were transfected with CaMKIIa or T286A, which lacksautonomy. Cell death was assessed by staining with, in this example,Ethidium Homodimer 2

In other correlative experiments, CaMKII overexpression was examined andwas found to enhance glutamate-induced neuronal cell death (see FIG.13B). Neuronal cell death by stroke (and traumatic brain injury, andneurodegenerative diseases) are thought to be caused mainly by“excitotoxcity” (toxic effect of excessive excitatory neurotransmitters,mainly glutamate). This observation is consistent with protectiveeffects observed after CaMKII inhibition. In addition, traditionalCaMKII inhibitor KN93 was examined KN93 protects from glutamate inducedneuronal death only when applied during but not after glutamate insult.This is in contrast to a construct of the instant invention, tatCN21,that not only protects during but was also demonstrated to protect afterglutamate insult (see FIGS. 11A and 11C). Both inhibitor tatCN21 and atraditional inhibitor (KN93) protected neurons from excitotoxic celldeath in culture, at least when present during the glutamate insult(cell death was reduce by half). Remarkably, tatCN21 (but not KN93) wasprotective also when added 1 h after the insult (and likely up to 4-6 hafter). Glutamate has at least four effects on CaMKII (inducing 2activation states, and 2 forms of translocation), and at least on isaffected differently by tatCN21 and KN93: tatCN21 (in contrast to KN93)blocks not only stimulated but also autonomous CaMKII activity(generated by T286 auto-phosphorylation). Thus, autonomous CaMKIIactivity is a drug target for therapeutic protection from excitotoxiccell death after an insult. These conclusions based on the inhibitorstudies were corroborated by overexpression of CaMKII wild type enhancedneuronal cell death; overexpression of the constitutively autonomousT286 mutant enhanced it even more, while the autonomy-incompetent T286Amutant had little or no effect.

Example 6

FIGS. 14A-14C illustrates a minimal inhibitory region of CaM-KIINretains CaMKII specificity. Three overlapping 21 amino acids longpeptides were derived from the previously identified inhibitory regionof CaM-KIIN (FIG. 1A). Their effect on AC2 substrate phosphorylation byCaMKII was then assayed in vitro (FIG. 1B). The N-terminal peptide CN21ashowed the full inhibitory effect observed with the full-lengthCaM-KIINtide (here named CN27). The other 21 mer peptides, CN21b andCN21c, had minimal or no effect. C-terminal truncations of CN21a by 4and 7 amino acids significantly impaired inhibition, although CN17astill clearly affected CaMKII activity (FIG. 1B). Thus, the fullinhibitory activity is contained in CN21a (CaM-KIIN 43-63). CN21a alsoblocked phosphorylation of crude liver protein extracts and ofbacterially expressed GST-NR2B-c (FIGS. 14A-14C). CN21a (5 μM) blocksCaMKII phosphorylation of (FIG. 14A) crude live protein extracts, (FIG.14B) bacterially expressed GST-NR2B-c(containing C-terminal NR2B aminoacid 1,120 to 1,482), and (FIG. 14C) T305 but not T286autophosphorylation. Phosphorylation was detected by ³²P autoradiography(FIG. 14A, 14B), or by phosphospecific antibodies (FIG. 14C). In FIGS.14A and 14B, CaMKII was autophosphorylated before the final reactions 13min at 30° C.) in order to reduce the T286 signal.

Example 7

FIGS. 15A-15D represent CN19, a minimal inhibitor region of CaM-KIIN.FIG. 15A represents a previously identified CaM-KIIN inhibitory region,CN21. Arrows indicate truncations that lead to reduced potency of CaMKIIinhibition. Truncation of the N-terminus by 2 amino acids completelyabolished CaMKII inhibition, and potency was significantly reduced evenby deleting only 1 amino acid. However, C-terminal truncation by 1 or 2amino acids did not affect potency. Only further truncation by 3 aminoacids significantly reduced potency (see FIG. 15C). Thus, theC-terminally truncated CN19 contains the minimal inhibitory region withfull inhibitory potency. Phosphorylation of syntide 2 by CaMKII wasinhibited by CN21, CN20 and CN19 with an IC50 of ˜100 nM (FIG. 15D).Syntide 2 is a “regular” S-site binding CaMKII substrate and CN peptidesinhibit its phosphorylation in a non-competitive manner. FIGS. 15B-15Drepresent that this particular CN19 retains full inhibitory potency,while any other further truncations are shown to reduce inhibitorypotency, as determined by the effect on CaMKII activity in a biochemicalassay of phosphate incorporation into the substrate peptides AC2 orsyntide 2, as indicated. It is contemplated that other reductions orsubstitution are considered in embodiments herein and that other aminoacid sequences of 19 or less amino acids can retain inhibitorycapabilities. It is also contemplated that certain amino acid sequencesof 21 amino acids or less in this region may be better suited fortherapeutic treatments than others. Bars grouped and labeled (**) do notdiffer from each other as determined by ANOVA. Error bars indicates.e.m. in all cases.

FIGS. 16A and 16B illustrate that tatCN21 dramatically reduces CaMKIactivity (**). Tat alone and other tat fusions had statisticallysignificant but much milder effects on CaMKI (*). CN27, 21, and 19 alonehad no significant effect on CaMKI activity (nd; ANOVA). FIG. 16Brepresents a dose response of CaMKI inhibition by tatCN21, tat, and CN21further demonstrates that CaMKI inhibition dramatically enhanced by thespecific fusion. Error bars indicate s.e.m. in all cases.

Example 8

In another exemplary method, a mutational analysis of CN19 was performedand effects of CN19 mutations were examined for CaMKII inhibition wasanalyzed. Truncation to a 19 mer enabled efficient small-scale peptidesynthesis in a 96-well format, and thus efficient generation of CN19mutation series. In an initial screen, each CN19 residue was substitutedindividually for Ala, and tested the effect on CaMKII inhibition. MostAla substitutions significantly reduced CN19 inhibition potency: sixmutants reduced potency more than 3-fold, and an additional eightmutants reduced potency to a lesser extent; only 2 mutants demonstratedno significant change (FIGS. 17A-17B). However, three mutants (P3A,K13A, and R14A) actually significantly increased potency of CN19 (FIG.2). CN19 contains 8 charged residues, and it was proposed that theseresidues might contribute significantly to interaction with andinhibition of CaMKII. However, only one charge substitution reducedpotency more than 3-fold was R11A, and two of the charge substitutionseven increased inhibition potency. Another surprising effect was thegreater than 3 fold decrease in potency by an S12A substitution, as theβ isoform of CaM-KIIN contains an Ala in this position, constituting theonly difference between the two isoforms within their inhibitory region.These random Ala scan substitutions provided results surprising resultsthat were not predictable. FIGS. 17A and 17B represent a summary of themutational analysis of CN19 and effects on potency of CaMKII inhibition.FIG. 17A represents that the first Ala scan of CN19 revealed severalmutations that significantly enhanced potency. Error bars indicates.e.m. FIG. 17B represents a summary of mutational analysis. Threemutations significantly increased potency; two additional ones slightly(the latter ones indicated in non-capital letter). Some specificmutations of S12 are (R, V), while most others are not. Several neutralsubstitutions for positive residues can be made; the maximal“combination” that remains neutral is the mutant “m1”.

In another example, mutations to positively charged amino acids wereexamined for a portion of the CaMKIINα, CN19. FIG. 18A represents dataof several point mutations to Arginine (R) that do not affect potency ofCaMKII inhibition identified using a CN19a3 mutant background. FIG. 18Brepresents the mutant CN19a2-m1 that contains a maximal combination ofpositively charged mutations without decreasing inhibitory potency.Error bars indicate s.e.m. in all cases. FIG. 18C illustrates additionalmutations made in the combination mutatant series, in addition to the3,14A mutations in CN19a2.

FIGS. 19A and 19B represent CN19 mutations at S12 and effects of thesemutations on CaMKII inhibition. FIG. 19A illustrates that most S12mutations were detrimental to potency of CaMKII inhibition in thecontext of the CN19a3 mutant (and CN19 wild type; see FIG. 17A, S12 to Aand D mutants). However, the S12 to V and R mutants had little effect,or even slightly increased potency of inhibition, respectively. FIG. 19Brepresents that the same findings were made for the S12 mutations in theCN19a2 background. Thus, S12R (and to some extend S12V) are viablemutations that can prevent phosphorylation of CN peptides within cells,thereby preventing inactivation.

FIGS. 20A-20C illustrate potency of combination mutants 1. FIG. 20Arepresents the combination mutants aX, a3, a4, and a5 that havesignificantly increased potency of CaMKII inhibition over CN19 wildtype. However, potency over the single mutant 13A is mildly increased,if at all, and potency over the single mutant 14A is dramaticallydecreased. The a3 mutant showed slightly better inhibition than the aXmutant, indicating that a 3,14A mutant (now called a2) might bebeneficial over 14A alone. FIG. 20B represents several additionalcombination mutants at higher concentration. The better inhibition of bya3 compared to aX (13/14A) was confirmed. FIG. 20C represents that P4appears to be unable to be mutated without reduction in potency, even ina background that contains a P3A mutation. Thus, it is likely thathelical structure at the CN19 N-terminus cannot be extended beyond the3^(rd) amino acid. Error bars indicate s.e.m. in all cases.

FIGS. 21A-21C represents potency of combination mutants II. (FIGS. 21Aand 21B) Dose response showed that potency of the CN19a3 mutant issignificantly increased over CN19 wild type. FIG. 21C represents that ana2 mutant has higher potency than the a3 mutant (predicted from FIG.20), as it shows significant inhibition even at 10 nM (in contrast toa3; see panel B). However, the single mutant 14A has even higherpotency, with an IC50 in the single digit nM range. Thus, its potency isincreased well over 10 fold compared to CN19 wild type. In context ofthe a2 mutant, mutations of position 19 to A or C were detrimental,while mutation to Y was not. Error bars indicate s.e.m. in all cases.

Example 9

CN19 phosphorylation at S12 by protein kinase C reduces potency ofCaMKII inhibition. In another exemplary method, protein kinase C (PKC)activity was examined with respect to CN19 as a substrate or the S12mutant. It was observed that S12 can be phosphorylated by PKC and mostbut not all S12 mutations decrease potency. The CN19 region mostsensitive to Ala substitutions (surrounding R11) contains a potentialPKC phosphorylation site (S12). CN19 but not its S12A mutant wasphosphorylated by PKC, with a phosphorylation rate of >50% compared to apeptide derived from a major PKC substrate protein (FIG. 22A) (usingMARCKS; myristylated alanin-rich C-kinase substrate). Additionally, thephospho-mimetic S12D mutation of CN19 significantly decreased potency ofCaMKII inhibition (FIG. 22B). This experiment demonstrates that CaMKIIinhibition by CaM-KIIN or CN peptides (e.g. CN19) can be modulated bycellular signaling leading to S12 phosphorylation. For example, thepost-translational modification can interfere with CaMKII inhibition. Inanother experiment, in order to prevent such inactivating effect whenusing CN peptides (e.g. as research tools or for modulating events in asubject) for CaMKII inhibition within cells, it may be desirable tosubstitute S12 with residue that cannot be phosphorylated. It wasdemonstrated that S12A reduced CN19 potency similar to S12D (FIG. 22B).Thus, alternative substitutions of S12 were examined. It was shown thatwhile S12 substitutions with amino acids G, P, N, F or L alsosignificantly reduced potency, substitution with V, R, or Q did notreduce potency (data not shown but see Table 1 and FIG. 23B).

FIGS. 22A and 22B represent CN19 phosphorylation at S12 by PKC reducespotency of CaMKII inhibition. A, PKC phosphorylated CN19 but not itsS12A mutant in vitro, as measured by phosphate incorporation. Thephosphorylation rate of CN19 was >50% compared to a MARCKS peptidesubstrate of PKC. B. Phospho-mimetic S12D mutation significantly reducedCN19 ability to inhibit CaMKII activity (measured by syntide 2phosphorylation in vitro). S12 was sensitive also to various othermutations (see Table 1 and data not shown). Error bars indicate s.e.m.in all panels.

Example 10

In another method, alternative substitution of S12 with other residueswas evaluated. Combining mutations to further increase CN19 potency wereevaluated. In order to improve potency of inhibitory effects of CNpeptides, combinations of three mutations that individually increasedpotency of CaMKII inhibition were examined. A triple mutant having aminoacid substitutions as follows: P3A, K13A, R14A (CN19a3) significantlyincreased potency compared to single mutant with the highest potency,R14A (FIG. 23A). It was demonstrated that the largest increase in CaMKIIinhibition potency was observed for the double mutant P3A, R14A (CN19a2)(FIG. 23A).

In addition, CN19a2 (with the double mutation) could be combined with anamino acid substitution, S12V (CN19a2v) to prevent S12 phosphorylationwithout loss of inhibition potency (FIG. 23B). It was demonstrated thatpotency of CN19a2v was significantly stronger compared to CN19a3 withadditional S12V mutation (CN19a3v) (FIG. 23B). It was demonstrated thatCN19a2v had an IC50 of ˜20 nM (FIG. 23B); the potency of CN19a2v wasabout 5-fold greater than CN19 (see FIG. 17A).

Several additional rounds of mutations (which mainly testedsubstitutions in CN19 wt, a2, or a3 with positively charged R or Kresidues) did not yield any further improvements over CN19a2v. However,several residues that could be substituted without reduction in potencywere identified (data not shown). This included positions 3 (K), 5 (R),7 (R,K), 8 (K), and 9 (L). The results, inferring the effects of alltested substitutions individually, are summarized in Table 1.

FIGS. 23A and 23B represent CN19a2v (SEQ ID NO: 23), a combinationmutant. Effect of CN19 peptides (20 nM) on CaMKII activity towardssyntide 2 in vitro was examined. A, CN19a2 (P3A, R14A, SEQ ID NO: 25)was the most potent double mutant of CN19, and also more potent that thetriple mutant CN19a3 (P3A, K13A, R14A) (**, p<0.01). B, S12V mutation ofCN19a2 (CN19a2v) did not reduce potency, and was more potent than a S12Vmutant of CN19a3 (***, no difference between a2 and a2v, but p<0.001compared to the other conditions). Error bars indicate s.e.m. in allpanels.

Example 11

In other exemplary methods additional strategies were implemented tofurther improve potency of CN19 to block CaMKII activity. Initial Alascans revealed that amino acid position R11 is the single most importantcharged residue for potency of CaMKII inhibition. Because a minimalconsensus sequence for CaMKII substrates is RxxS/T, it was reasoned thatR11 might provide the R at the −3 position for a pseudo-substratesequence within CN19 that directly blocks the substrate binding site(see for example FIG. 24A). Thus, several mutations were examined thatmake the CN19a2v region around R11 closer to an optimal CaMKII substratesequence (without introducing a phosphorylatable residue to the peptide)(FIG. 24A): Although less important than the R at the −3 position,CaMKII has been determined to prefer substrates with hydrophobicresidues at the −5 and +1 positions and also shows some preference forsubstrates with Q at −2, F at +1 and D at +2 positions. Both V12Q andV16D have been demonstrated to significantly increase potency of CN19(see for example FIG. 24B). V15I also increased potency, however, theV15F mutation in this position did not (FIG. 24B). The V12Q and V16Dmutants of CN19a2v had an IC50 of about 1 nM, and a combination mutant(referred to as CN19o) increased potency even further (FIG. 24C). 5 nMCN19o blocked CaMKII activity nearly completely (supplemental FIG. S6).Additional combination of CN19o with V15I or V15F did not furtherincrease potency of inhibition (FIG. 24C).

FIGS. 24A to 24C represent how another approach to mutagenesisdramatically improved potency in the peptide, CN19o (SEQ ID NO: 24). A,Sequence alignment illustrates the rational for further mutagenesis. Theinitial Ala scan indicated that R11 of CN19 may constitute the −3position R of a pseudo-substrate sequence. B, The CN19a2v mutationsV12Q, V15I and V16D dramatically increase CaMKII inhibition. C, Theoptimal combination mutant of CN19a2v was V12Q, V16D (CN19o). AdditionalV15F mutation did not further increase inhibition of CaMKII (0.5 nM),and all other combination mutants showed less CaMKII inhibition (**,p<0.05). Error bars indicate s.e.m. in all panels.

Example 12

It was unexpected when it was observed that CN19o (SEQ ID NO: 24) hasboth highly increased potency, as well as, selectivity and may be theoptimum peptide for selective inhibition of CaMKII activity. CN19o (SEQID NO: 24) inhibited CaMKII with an IC50 of about 0.75 nM (FIG. 25A),and thus CN19o (SEQ ID NO: 24) has >125 fold enhanced potency comparedto CN19. If any, this result underestimates the potency of CN19o (SEQ IDNO: 24): Even though concentration of CaMKII in this assay wassignificantly reduced compared to standard assays (to 0.1 nM), theinhibitor excess at the determined IC50 was only 7.5 fold.

Further investigation revealed that CaMKI inhibition by CN19o (SEQ IDNO: 24) (about 38 μM IC50) was reduced compared to CN19a2v (about 18 μMIC50). CaMKI inhibition by CN19o (SEQ ID NO: 24) was demonstrated to besimilar to CN19 (about 36 μM IC50) (FIG. 26). Thus, the resultingselectivity of CN19o for CaMKII vs CaMKI is about 50,000 fold (about125-fold greater than other peptides disclosed herein).

Additionally, CN19o was tested for effects on a panel of other kinases(see for example FIG. 25B). This panel included other CaM kinase familymembers (CaMKIV, DAPK1, AMPK) and other basophilic multifunctionalkinases (protein kinase A (PKA) and protein kinase C (PKC)). Whenincreased to 5 μM (>6,500 fold IC50 for CaMKII), CN19o (SEQ ID NO: 24)did not significantly affect activity of these other kinases tested.Thus, CN19o (SEQ ID NO: 24) inhibits CaMKII both with remarkable potencyand high selectivity. Certain embodiments disclosed herein may bedirected at using a highly potent inhibitor of CaMKII (e.g. CN19o) or aless potent inhibitor of CaMKII depending on the circumstances and theneed.

FIGS. 25A and 25B represent increased potency and selectivity of CN19o(SEQ ID NO: 24). A, The IC50 for CaMKII inhibition by CN19o (SEQ ID NO:24) is about 0.75 nM, as indicated by CaMKII activity assays. CaMKIIconcentration was lowered to 0.1 nM for CN19o (SEQ ID NO: 24)concentration below 5 nM. B, While 5 nM CN19o (SEQ ID NO: 24) completelyblocked CaMKII activity, even 5 μM CN19o (>6,500 fold IC50 of CaMKIIinhibition) did not significantly affect kinase activity of CaMKI,CaMKIV, DAPK1, AMPK, PKA, or PKC. Error bars indicate s.e.m. in allpanels.

FIG. 26 represents CN19, CN19a2v, and CN19o inhibition of CaMKI, whichwere observed to be 360-50,000 fold less potency compared to inhibitionof CaMKII. The IC50 for CaMKI inhibition of CN19 (36 mM), CN19a2v (18mM) and CN19o (38 mM) 360-50,000-fold greater than their IC50 for CaMKIIinhibition (100 nM, 20 nM and 0.75 nM, respectively; compare FIGS. 15,23 and 26). For curve fit, the Hill coefficient was set as 1.

TABLE 1 Effects of individual CN19 mutations on CaMKII inhibitionsummary and improvements over certain previously examined mutants CN19 +~ −  1 K AR  2 R A  3 P A ~K > R  4 P A K  5 K R AR  6 L A  7 G RK R  8Q A K R  9 I L AR 10 G A 11 R A 12 S Q * VR A KD GPNFL 13 K A 14 R A 15V (I)* AR(F)* 16 V D * A R 17 I ARK 18 E AR 19 D AR C Mutations thatwere combined for the optimized CN19o are marked in bold and underlinedin the second column. Asterisks indicate the rational mutagenesis.

Materials and Methods

Peptides and proteins. CaMKIIα and β were purified from abaculovirus/Sf9 cell expression system, CaM was purified after bacterialexpression (as presented previously).2-chloro-(ε-amino-Lys₇₅)-[6-(4-N,N-diethylaminophenyl)-1,3,5-triazin-4-yl]calmodulin(TA-CaM) was obtained. GFP-CaMKIIα wild type and mutants were expressedin Cos-7 cells and extracts were prepared in 50 mM PIPES, pH 7.2, 10%glycerol, 1 mM EGTA, 1 mM DTT, and protease inhibitors (BoehringerMannheim). 0.2 g/ml rat liver was homogenized in the same buffer andspun for 10 min at 10,000 g. GST-NR2B-c (amino acids 1,120-1,482 of thecytoplasmic NR2B C-terminus) was expressed in bacteria, previouslydescribed. CaM-KIIN, MAP2, AC2, syntide2 (Sigma), calmodulin bindingdomain (CBD; Calbiochem), tat fusion peptides (Global Peptides), andother CN peptides (Caltech Synthesis Core) were obtained.

CaMKII activity assays. Standard CaMKII assays were done for 1 min at30° C. previously described, with 20 nM CaMKII (subunit, not holoenzymeconcentration), 50 mM PIPES pH 7.2, 0.1 mg/ml BSA, 10 mM MgCl₂, 100 μM[γ-³²P] ATP (˜1 Ci/mmol), 1 mM CaCl₂, 1-2 μM CaM, and 30-60 μM AC2peptide (or syntide2). The reactions were spotted onto Whatman P81phosphocellulose paper rectangles (˜2×2.5 cm). To remove freeradioactivity, the paper rectangles were rinsed and washed for 30 minunder agitation in 0.5% phosphoric acid or water. After two more rinses,an additional 30 min wash typically did not release any more measurableradioactivity. Radioactivity of the bound peptides was quantified in aBeckman 6000TA scintillation counter by the Cherenkov method. Anychanges of the standard protocol were done as indicated. For assays ofthe GFP-CaMKIIα mutants, kinase amounts were normalized by GFPfluorescence in the extract, and total protein was adjusted with Cos-7extracts.

Kinase panels. A panel of different kinases was tested utilizing akinase profiling service (Upstate Biotechnology). 40 min reactions atroom temperature contained 0.1% BSA and were started by addition of 10mM MgAcetate and [γ-³³P-ATP], stopped by 0.1% phosphoryic acid, spottedon filtermats, and washed 3×5 min in 75 mM phosphoric acid and 1× inmethanol prior to drying and scintillation counting. CaMKII and IV wereactivated by 5 mM CaCl₂ and 1.7 μM CaM in 40 mM Hepes pH 7.4; substratewas 30 μM KKLNRTLSVA (SEQ ID NO:22). Buffers and substrates for theother kinases are stated in brackets: PKA (8 mM MOPS pH 7, 0.2 mM EDTA;30 μM Kemptide), PKCα (20 mM Hepes pH 7.2, 0.3% Triton X-100, 0.1 mg/mlphosphatidylserine, 10 μg/ml diacylglycerol; 0.1 mg/ml histone H1),JNK1α1 (50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercapto-ethanol; 3 μMATF), MAPK1 and raf (25 mM Tris, pH 7.5, 0.02 mM EGTA; 250 μMproprietary substrate and 0.66 mg/ml myelin basic protein,respectively). Reactions in presence of 5 μM CN21a were done induplicate, and normalized to four parallel reactions without inhibitor.

CaMKII auto- and protein-phosphorylation. 10 nM MAP2 (˜140 nMphosphorylation sites) was used instead of substrate peptide, andreaction times were 5 min, unless indicated otherwise. CaMKIIconcentration was 100 nM subunits (=8.3 nM holoenzymes). Auto- andMAP2-phosphorylation were assessed by Immuno-blot analysis previouslyderived (Bayer et al., 2002, incorporated herein by reference) withphospho-T286- or -T305-specific antibodies (1:500; PhosphoSolutions) andan anti-phospho-Thr antibody (1:500; Zymed), respectively. Total CaMKIIαwas detected with CBα2 antibody (1:2000; GIBCO). Protein were separatedon 10% poly-acrylamid SDS Gels, and electro-blotted onto “Protran” 0.2μm pore nitrocelluse filters (e.g. Schleicher & Schull) or onto PVDFfilters (e.g. Perkin Elmer). Alternatively, for quantitative analyses, avacuum-driven slot-blot manifold (e.g. Schleicher & Schull) was used fortransfer onto PVDF membranes. PVDF membranes were air-dried for 15 min,then wetted in Methanol. Blots were blocked in 5% milk in TBS-T(Tris-buffered saline pH 7.6 with 0.1% Tween-20). Antibodies wereincubated for 45-60 min at room temperature in 2.5% milk in TBS-T; forthe anti-phospho-T305 antibody, 2.5% BSA was used instead. Aftersecondary antibody incubation (anti-mouse or anti-rabbit horseradishperoxidase conjugate; Amersham; 1:4000), detection was done using the“Western Lightning” system (Perkin Elmer) and exposure to “Hyperfilm”(Amersham). For quantitative analysis, chemoluminescence was capturedusing a Chemilmager 4400 imaging system (Alpha Innotek) instead of film.Only non-saturated images were analyzed, using AlphaEase software.

Crude liver extracts or NR2B-c were phosphorylated with 100 nMautophosphorylated CaMKII, 2 μM CaM and 40 μM [γ-³²P]-ATP (˜2 Ci/mmole).Pre-auto-phosphorylation (of 300 nM CaMKII) was done for 5 min on ice,in presence of 3 mM CaCl₂, 6 μM CaM and 120 μM unlabelled ATP. Afteraddition of [γ-³²P]-ATP and inhibitors as indicated, 2 min reactions at30° C. were started by addition of substrate protein extract (⅓^(rd) to1/12^(th) of total reaction volume), and stopped with 25 mM EDTA.Phosphorylation was detected by autoradiography after gelelectrophoresis.

CaMKII binding to NR2B-c was assessed as previously described. Briefly,GST-NR2B-c was immobilized on anti-GST coated microtiter plates, thenoverlaid with 100 nM CaMKII in presence of Ca²⁺/CaM and 1 or 5 μM ofvarious CN peptides. After extensive wash, protein was eluted from theplates by boiling in SDS-loading buffer. Eluted CaMKII was detected byWestern-blot as described above.

TA-CaM dissociation was assessed by increased TA-CaM (30 nM)fluorescence after dissociation from CaMKII or its CaM-binding domain(CBD) (150 nM) during a chase with unlabeled CaM (60 μM). Buffercontained 50 mM Hepes pH 7.4, 150 mM KCl, 2 mM MgCl₂, 2 mM MgADP, 2 mMCaCl₂, and 0.1 mg/ml BSA. Fluorescence was measured in a time scan (1sec samples) at 365 nm excitation and 415 nm emission wavelength on aspectro-fluorometer (Fluorolog3; Horiba Jobin Yvon) and was correctedfor photobleach (FIG. 16).

Imaging of neuronal filopodia motility was done similarly as previouslydescribed. Hippocampal cultures were prepared from newborn SpragueDawley rats (Harlan) as previously described, plated onto poly-D-lysinecoated glass bottom dishes (MatTek) at a density of ˜2.5×10⁶ cells/cm²and maintained in Neurobasal A medium with penicillin/streptomycin (50units/ml), glutamax (2 mM) and B27 supplement (Invitrogen). Glial growthwas inhibited by 5-fluoro-2′-deoxy-uridine and uridine (70 μM and 140μM). After 5 days in vitro, neurons were transfected with a GFPexpression construct (Clonetech) using lipofectamine 2000 (Invitrogen),as described. On the next day neurons were imaged in culture medium onfor example, a Zeiss Axiovert 200M systems equipped with a 40× oilimmersion objective, Cool Snap HQ CCD camera (Roeper Scientific), Xenonlamp LB-LS/17 (Sutter Instruments) and climate control set to 30° C. and5% CO₂. Fluorescence Images were acquired and analyzed using SlideBooksoftware (Intelligent Imaging Innovations). 16 images were taken in 20 stime intervals, at 100 ms exposure time and bin factor 2. Subtractionimage (A image) stacks were generated by subtracting one stack (firstimage deleted) from its duplicate (last image deleted). Then, A imagestacks were converted into average images in pseudocolor, for bettervisualization of motility. Quantification of the pixel intensity yieldeda relative motility index, expressed as A image intensity beforetreatment set as 100%. Intensity cutoff masks eliminated most backgroundpixels not located within neurons. Neurons were imaged before and after20 min incubation with either tatCN21 or tatRev (5 μM). Incubation wasdone on the imaging setup; data from experimental days on which mockincubation without peptide affected motility were discarded.

Insulin secretion. Langerhans' islets acutely isolated from adult maleWistar rats (Harlan) were obtained. On 24-well plates, 10 islets werepooled per well in 20 mM Hepes, 25 mM NaHCO₃, 114 mM NaCl, 4.7 mM KCl,1.2 mM KH₂PO₄, 1.16 mM Mg50₄, 2.5 mM CaCl₂, 0.2% BSA; adjusted to pH7.2. Within 90 min after isolation, insulin secretion was stimulatedwith 11 mM glucose. Inhibitors or EGTA were added 30 min beforestimulation. Insulin secreted into the medium during 90 min stimulationwas measured using an ELISA kit (CrystalChem). Two independent isletpreparations showed inhibition of glucose-stimulated insulin secretionby tatCN21.

Statistics—All pair-wise comparisons were done using a two-tailedt-test, using Excel software (Microsoft). Comparisons of multipleconditions were done by one-way ANOVA with Newman-Keuls multiplecomparison post hoc analysis, using for example, Prism software(GraphPad).

All of the COMPOSITIONS and/or METHODS disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variation may be applied tothe COMPOSITIONS and/or METHODS described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itwill be apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A composition comprising an amino acid sequence comprising SEQ IDNO:23 wherein the amino acid sequence at positions 3 and 14 of SEQ IDNO:23 include an alanine; and a valine at position 12 to improve potencyof CaMKII inhibition compared to a control sequence.
 2. The compositionof claim 1, further comprising a cell-transfer/penetrating agentassociated with the amino acid sequence.
 3. The composition of claim 2,wherein the cell-transfer/penetrating agent is fused to the amino acidsequence.
 4. The composition of claim 1, wherein thecell-transfer/penetrating agent comprises tat, ant, meristyl-group,palmityl-group or combination thereof.
 5. The composition of claim 1,further comprising a pharmaceutically acceptable derivative or saltthereof.
 6. The composition of claim 1, further comprising one or moreagents for treating a neurodegenerative condition.
 7. The composition ofclaim 1, further comprising one or more mutations in the amino acidsequence.
 8. A composition comprising an amino acid sequence comprisingSEQ ID NO:24 wherein the amino acid sequence positions 3 and 14 of SEQID NO:24 are alanine, a glutamine at position 12 and an asparagines atposition 16 to improve potency of CaMKII inhibition compared to acontrol sequence, SEQ. ID NO:1.
 9. The composition of claim 8, whereinone or more additional amino acids are substituted for arginine,alanine, valine, lysine or combination thereof.
 10. A method fortreating a subject having or suspected of developing a condition causingneuronal cell death comprising: administering to a subject in needthereof a therapeutically effective amount of a composition comprisingSEQ ID NO:24.
 11. The method of claim 10, further comprising acell-transfer/penetrating agent associated with the amino acid sequence.12. The method of claim 10, wherein the condition causing neuronal celldeath comprises stroke, ischemia, traumatic brain injury, orneurodegenerative disorders.
 13. The method of claim 10, furthercomprising administering one or more additional treatments forinhibiting neuronal cell death to the subject.
 14. A method for treatinga subject having or suspected of developing a drug addiction comprising:administering to a subject in need thereof a therapeutically effectiveamount of a composition comprising SEQ ID NO:24.
 15. The method of claim14, further comprising a cell-transfer/penetrating agent associated withthe amino acid sequence.
 16. A method for treating a subject having atraumatic brain injury comprising: administering to a subject atherapeutically effective amount of a composition comprising SEQ IDNO:24.
 17. The method of claim 16, wherein the traumatic brain injurycomprises stroke, ischemia, Alzheimer's disease, Parkinson's disease,spinal cord injury, or drug addiction.
 18. The method of claim 16,further comprising administering one or more additional treatments tothe subject directed to treating the traumatic brain injury.
 19. A kitcomprising: a vessel; and one or more composition comprising an aminoacid comprising SEQ ID NO:24 wherein the amino acid sequence positions 3and 14 of SEQ ID NO:24 are alanine, a glutamine at position 12 and anasparagines at position
 16. 20. The kit of claim 19, further comprisinga delivery device for administering the one or more compositions to asubject.